In 1938, President Franklin D. Roosevelt signed the Fair Labor Standards Act, which created a right to overtime pay for those who worked more than 40 hours a week.

Edward Teach (alternatively spelled Edward Thatch, c. 1680 – 22 November 1718), better known as Blackbeard, was an English pirate who operated around the West Indies and the eastern coast of Britain's North American colonies.

Hubert Czerniak TV - Fakty i mity o witaminie D3! Lepiej usiądźcie -

Witamina D3 + K2 MK-7 - 5.000 IU + 200mcg - 120szt.

Leczenie doustne: dorośli z niedoborem (<20 ng/ml 25(OH)D3): 40 000 IU/tydzień przez 7 tyg. lub 50 000 IU/tydzień przez 6 tyg.[4

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Дикий шок первого дня.Безумная неделя в коридоре, крики с крыш «Точно свои? Сейчас гранату кинем!», закрытые магазины и бесплатный хлеб.Выезд снежным утром, пустой город и бесконечная очередь машин, надписи «Дети» и брошенные на обочинах Теслы.Бесконечно холодные март и апрель за Днепром. Попытки собрать свой мир обратно из маленьких кусков.Возвращение в пустой и грохочущий Харьков. Безлюдные проспекты, надписи «Закрыто, ничего нет», следы от прилетов.Белгородский экспресс в 10, возвращение девочек из Германии, привычку к горящему горизонту и взрывам за окном.Сентябрь 2022, когда фронт отодвинули от Харькова, непривычную тишину и улыбки на лицах.Момент, когда мы отмечаем компанией снятие осады — и после тяжелого взрыва за окном гаснет свет. На сутки. Полгода блекаутов и жизни с батарейками.«Дальше будет только лучше, войне скоро конец!» Бравые записи в каналах и новостях, намеки от властей и командующих. Вирусные открыточки и мемчики.Разочарование лета 2023. Ничего не закончилось.

A typewriter repair technician by trade from Michigan, Carl Elmer Anderson started the Anderson Typewriter Company in Pasadena in 1912 after falling in love with the City as a vacationer.

every person who got apnea got to be on vitamin d vitamin d makes a tremendous change for so many different 00:24:20 chemical changes in our entire body uh it affects you not only our immune system because you know we look at covet they you know they found like eight over eighty percent of every covert case had deficiency of vitamin d so you think about if your immune system 00:24:33 is weakened that means other systems are weakened which what does the immune system do it keeps away inflammation you see it's all tied together it's so important so now you say why do i need vitamin d well how much are you in the 00:24:46 sun as we get older we're just not in the sun all that much and oh i eat healthy you're not getting it from you're not from your food you need to supplement

Data and analytics engineers for Gentrack Logical Data Model

In 1950 Dwight Eisenhower wrote a letter to a U.S. diplomat in which he ascribed a military-oriented version of the saying to an anonymous soldier. Emphasis added to excerpts by QI:[1]1984, The Papers of Dwight David Eisenhower, Volume XI: Columbia University, Editor Louis Galambos et al, Letter from: Dwight Eisenhower, Letter to: Hamilton Fish Armstrong, Date: December 31, 1950, … Continue reading . . . I always remember the observation of a very successful soldier who said, “Peace-time plans are of no particular value, but peace-time planning is indispensable.”

Dr. Martin Luther King, Jr.

my peers, stared, their faces as unconvinced as blank sheets of paper.

«В Японии бывало много религиозных споров и, между прочим однажды о том, какого цвету диавол: одна секта утверждала, что он черный как уголь, другая, напротив, что у него кожа снежной белизны; но тут возникли еще две партии и, увлекаясь духом противоречия, стали утверждать, одна что он красный, другая, что зеленый. Распря разгоралась, умы воспламенялись, и чуть не дошло до междоусобной войны. Наконец дело представлено было на разрешение императора, и сын неба помирил всех, объявив, что все правы. С тех пор японский ад населился чертями черными, белыми, красными и зелеными, народ перестал спорить и с равным удовольствием смотрит на тех и других, любуясь их пестротою и разнообразием».

As socialist realism was imposed on Soviet writers, one form of permissible resistance, of finding an inner freedom, was to read translations of foreign writers. No private library was complete without Hemingway, Faulkner, London, Fitzgerald, Steinbeck, Salinger—all officially permitted as “progressive writers” exposing the “ulcers of the capitalist world.”

Or, did you ever see a dog with a marrowbone in his mouth,—the beast of all other, says Plato, lib. 2, de Republica, the most philosophical? If you have seen him, you might have remarked with what devotion and circumspectness he wards and watcheth it: with what care he keeps it: how fervently he holds it: how prudently he gobbets it: with what affection he breaks it: and with what diligence he sucks it. To what end all this? What moveth him to take all these pains? What are the hopes of his labour? What doth he expect to reap thereby? Nothing but a little marrow

Cohen was reluctant to provide reference values for his standardized effect size measures. Although he stated that d = 0.2, 0.5 and 0.8 correspond to small, medium and large effects, he specified that these values provide a conventional frame of reference which is recommended when no other basis is available.

Justice Louis D.Brandeis instructedlawyers that “there is nosuch thing as good writ-ing. There is only goodrewriting.”23

Additionally, Henry Giroux claims that it was originally "military–industrial–academic complex".[16]

Одна из лучших возможностей нашего времени, кроме стоматологии с обезболиванием — возможность всегда с кем-то поговорить о чем угодно

Set frequency to 145.670

У нас в стране происходит самая тяжелая гуманитарная катастрофа последних 80 лет, во всех смыслах, и что же мы видим, или вернее не видим?Я встречал гуманитарные склады и кухни в спортзалах, подвалах офисов, школьных столовых, конюшнях и моллах.Но никогда не видел и не слышал, чтобы они были в церквях или монастырях.Центры эвакуации? Бесплатная раздача еды? Центры психологической поддержки? Координаторы волонтеров? Организация поставок через границу, черт возьми?Кто угодно, только не товарищи в рясах.Такое ощущение, что их нет, и все эти огромные халабуды стоят брошенными именно в тот момент, когда они понадобились.Честно говоря, я не представляю, как такое может быть, но факт остается фактом. Церковь эту войну проиграла начисто, независимо от того, кому она подчиняется.- я вижу не так мало священников, которые пытаются что-то делать и часто успешно. Но какой-то организации, за ними стоящей, я не вижу: все разрозенно и держится на подвиге отдельных людей.Так что церкви как организации проигрывают несмотря на то что кто-то определенно будет прославлен в лике святых, явно или неявно.

Те, с кем я общался после начала войны, потухли. Буквально. Все, что они знали и любили, весь старый Харьков потерял смысл.Как можно после февраля рассказывать о том, как по приказу из Москвы строили Госпром? Или про то, как наши проходчики потом поехали делать метро в тогдашнюю столицу? Как делали Т-34 в прошлую войну?Вся, вся старая история бессмысленна, ее теперь невозможно рассказывать. Это не запрещено — просто невозможно. Она отошла, осталась где-то там, в нулевых. И ее знатоки вместе с ней.Прошлое время прошло.Гамлет, Розенфельд, Комаровский. Бушвокер.А новая история только пишется и новый город только возникает.И никто пока не может сказать какой он будет.Точно другой....И это еще не начали ломать, еще не упал Пушкин в начале бывшей Немецкой.

D*nald Trump

Oh, never, surely, was there knight So served by hand of dame, As served was he, Don Quixote hight, When from his town he came; With maidens waiting on himself, Princesses on his hack—

Ce programme nommé « I can word it too », disponible en hébreu et arabe, a été spécialement créé pour cette étude. Il reproduit les activités quotidiennes (jouer à des jeux, prendre les repas, faire sa toilette…) et demande à l’enfant ce à quoi il veut jouer, en lui présentant un choix de jeux sur l’écran

Make Use of Frames FacebookLinkedInTwitterPinterestEmailPrint Using frames or templates is a great way to scaffold instruction and build learners’ confidence in writing, particularly in writing tasks and genres with which they have little prior experience. A writing frame consists of a skeleton outline given to learners to scaffold their writing. By providing a few sentence starters and some rhetorical phrases common to the task or genre, frames give learners a structure that allows them to focus on expressing their thoughts

Depuis longtemps, je suis d’avis que la rigueur d’un cours ne se mesure pas à la quantité de connaissances dont l’enseignant fait étalage, mais aux apprentissages que les étudiants font.

PTH

Miscible with alcohol

(+)-Limonene is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide. The solubility of (+)-Limonene in these solvents is approximately 20 mg/ml.

les directeurs d'école et les chefs d'établissement doivent communiquer aux associations de parents d'élèves qui en font la demande la liste des parents d'élèves de l'école ou de l'établissement scolaire mentionnant leurs noms et adresses postale et électronique

108,574

The more coachable someone is, the more they can grow, and the more quickly they can grow. Someone with low coachability can find it so hard to do anything outside of their expertise that it is understandable when managers focus their energy on the people they can help and grow instead.

Empower managers to facilitate effective learning transfer As Fergal explains, managers have a key role to play in facilitating effective learning transfer. “Research shows that managers play the most critical role in learning transfer - especially in the post-training environment. Every learner needs a manager who understands them, and how they want to learn and grow. They need to have the right coaching style, and they need the right resources.”In most organizations, instructional design focuses on the needs of the learner. But as Fergal explains, focusing on the needs of your managers can pay dividends. “Ideally, you’d have the manager attend the same training as the learner. The problem is, managers are always stretched. So, what you can do instead is develop specific guidance for your managers.” Provide a script for managers to support their team’s learning

Your employee experience action plan Delivering a more positive experience for employees begins with diagnosis – listening to the voice of your employees frequently and consistently through the power of tools such as pulse surveys. It continues by identifying the culturally relevant workplace practices that you can build on and improve. Once that’s done, it’s time to take action:  Enable managers to design experiences consistent with your organization’s core values

Why do 87% of data science projects never make it into production?

Following are strategies for facilitating SDL. The teacher can help the learner to Conduct a self-assessment of skill levels and needs to determine appropriate learning objectives. Identify the starting point for a learning project. Match appropriate resources (books, articles, content experts) and methods (Internet searches, lectures, electronic discussion groups) to the learning goal. Negotiate a learning contract that sets learning goals, strategies, and evaluation criteria. Acquire strategies for decision-making and self-evaluation of work. Develop positive attitudes and independence relative to self-directed learning. Reflect on what he/she is learning.

What Are the Differences Between Facilitation, Presentation, and Training? Trainers help others improve their performance by teaching, instructing, or facilitating learning. As such, facilitation and presentation are both tools in a trainer’s toolkit. In most cases, effective and engaging trainers will spend less time presenting content through lectures or lecturettes and more time facilitating learning around that content. Presentation vs. Facilitation

Learning is not linear, and meaningful learning resists being quantified. Our assessment approaches should create space for learning not arbitrarily delimit it.

The best course of action is to be intentional and systematic from the get-go. That is, rather than seeing the development, communication, and management of knowledge as “nice-to-haves” within your organization, start building these processes into your organization’s standard routine: Include knowledge management in your project descriptions and timelines. Set a “shelf life” for your knowledge documents (the maximum amount of time that can pass before revisiting said documents in some way). Perform scheduled maintenance to your knowledge documentation on a monthly, quarterly, and yearly basis.

MediaWi

A crucial difference between representations of relative error inthese equations compared withEquations 6and7 for the single-facet designs is that three sources of measurement error varianceare separately represented, withpt2ntequaling specific-factor error,po2noequaling transient error, andpto,e2ntnoequaling random-responseerror. Effects fortasks, occasions, and their interaction are includedin the denominator for the D-coefficient but not the G-coefficientbecause those effects can change the absolutemagnitude of scoresbut not their relative differences.

Most C++ template idioms will carry over to D without alteration, but D adds some additional functionality

Create an /etc/apt/preferences.d/debian-chromium

Learners’ practices, for example, should develop from low-level procedures such as sharing initial ideas to mid-level comparing and evaluatingsequences and eventually toward high-level challenging/debating and synthesizingroutines.

identify invariant (i.e., constant) characteristics of subsequent transfer domains

The freedesktop.org project also developed a free and open-source software library called libdbus, as a reference implementation of the specification. This library should not be confused with D-Bus itself, as other implementations of the D-Bus specification also exist

relief

Cholesterol

article D. 111-9 du code de l’éducation

À cette fin, comme le prévoit l'article D. 111-8 du Code de l'éducation, les directeurs d'école et les chefs d'établissement doivent communiquer aux associations de parents d'élèves qui en font la demande la liste des parents d'élèves de l'école ou de l'établissement scolaire mentionnant leurs noms, adresses postale et électronique, à la condition que ceux-ci aient donné leur accord exprès à cette communication.

Reverse engineering a bronze cannon from theLaBelleshipwreck

More girls

tamper-proof qubit

Overnight grown primary culture of E. coli cells (1 % v/v final concentration) was inoculated into 1 litre of LB media containing antibiotics. Culture was incubated at 37 oc at 200 rpm. Growth was monitored by measuring absorbance of E. coli broth at 600 nm. Culture was induced by adding 1 mM IPTG at an OD of 0.6 and was harvested after 4 hrs of induction. Samples were taken on an hourly basis after induction to check the kinetics of protein expression. Un-induced and induced E. coli cells were analyzed by SDS-PAGE to check the expression of recombinant protein.

Primary culture of E. coli was grown in LB medium containing either ampicillin (Amp) and/or kanamycin (Kan) to final concentration of 100 j..tg/ml and 25 J..tg/ml respectively. Depending on the vector construct, antibiotics were used for expression of different proteins as described in Table 3.1. Medium was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37 oc at 200 rpm.

The DFD between an atom pair is the normalized frequency distribution of the interatomic distances sampled from equal time snapshots taken from the MD simulations. DFDs of corresponding atoms taken from the different simulations were used to qualitatively compare the effect of mutational perturbations on the HbS fiber. Considering that the fiber simulations were carried out only for a relatively short time scale of 1.2ns, the calculated DFDs might suffer from errors due to limited sampling. Hence a quantitative comparison of the different DFDs were not attempted, however given that the global parameters monitored during the simulation had already become reasonably stable after 0.2ns (Chapter!, Table 5), it is expected that the gross features of the DFDs would remain unaltered even in much longer simulations.

The fluctuation maps (Fu) were calculated from the MD trajectories of the 0-chains as described earlier (Hery et al, 1997). The fluctuation value Fy is given by the equation, where dy(t) is the distance between a pair of designated atoms ( ca atoms as used here) at time t and the angle brackets represent time averages. The Fy values are the standard deviation of interatomic distance. The fluctuation maps in Figure 6 has a black dot wherever the Fu value is less than or equal to 0.5A. Thus dark regions of the map indicate those parts of the molecule which undergo strongly coupled movements.

chains were generated in the same way as for the isolated a-chains. Thus the model consisted of eight polypeptide chains ( 4 a-chains and 4 13-chains) with the central two a and two 13 chains making up the axial contact interface. The simulations of the above fiber models were carried out without explicit solvent using the GROMOS96 vacuum force field as implemented within the GROMACS suite of programs. Models of HbS mutants were generated using the program SCWRL and the mutant fiber models were generated as for the native HbS model. SCWRL replaces only the side chains of desired residues with the best possible rotamer of the mutated amino acids. Thus the initial backbone conformation of the isolated a-chains and the fiber models remained identical in the native HbS and the mutants. The MD simulation protocol for the fiber models consisted of an initial steepest descent energy minimization for 1000 steps followed by full MD simulation for 1.2ns at 300 K. Essential parameters of the simulation like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies are summarized in Chapter I, Table 4. It was observed that all the global indicators of the simulation stabilized to their average values within 0.2ns. Hence data from 0.2ns till the end of the simulations were used for all subsequent analysis

MD simulations on the a-chain of HbS and the mutants were carried out using the GROMACS suite of programs (Lindahl eta!, 2001). The initial structure of the native protein was taken from the high-resolution x-ray crystal· structure (Harrington et a!, 1997) of HbS (PDB entry: 2HBS). The structures of the mutants were generated interactively using INSIGHT-II. The initial model structures were placed in a simulation box of size 42.8 x 31.5 x 42.7 A. The closest distance from any protein atom to the walls of the box was not less than 9 A. The system was then solvated by adding a bath of SPC (Berendson eta!, 1981) waters in such a way that the density of the system was as close to 1 as possible. The overall charge of the system was neutralized by placing suitable counter ions wherever necessary (Chapter 1, Table 4). The resulting system was then energy minimized for 1000 steps using the steepest descent algorithm. This was followed by 0.3ns of position restrained MD during which the solvent and counter ions were allowed to move freely but the protein atoms were harmonically restrained to their initial positions. Finally, normal MD was run for 3ns using the default GROMACS force field. Bond lengths were restrained to their equilibrium values using the LINCS (Hess et a!, 1997) algorithm and a cut-off radius of 0.9nm was used for non-bonded interaction calculation. The temperature of the system was maintained close to 300K by weak coupling to an external temperature bath with a coupling constant of 0.1 ps. The integration time step used throughout the simulation was 1 fs. MD simulations of single mutant a-chains (K 16Q, E23Q and H20Q) as well as the double mutants (K16Q/H20Q, K16Q/E23Q and H20Q/E23Q) were carried out in a similar fashion for 3ns. In order to directly analyze the effects of the mutations on the contact interface, we also carried out MD simulations on a miniature model of the sickle hemoglobin fiber consisting of a complex of two hemoglobin tetramers. In the low salt crystal structure of deoxyhemoglobin S (Harrington et a!, 1997) the asymmetric unit consists of two HbS tetramers that pack as two strands of HbS molecules running parallel to the crystallographic a axis. Axial contacts occur between two tetramers of the same strand and lateral contacts occur between tetramers of different strands. A canonical fiber model was generated by taking one of the two tetramers in the asymmetric unit together with its neighbor translated along the crystallographic a axis [Figure 7 (A, B)]. Fiber models incorporating mutant a

Electrostatic potentials were calculated by the Finite Difference Poisson-Boltzmann (FDPB) method using the program MEAD running within the PCE web server (http://bioserv.rpbs.jussieu.fr/PCE) (Miteva et al, 2005; Bashford et al, 1992). Additions of hydrogen atoms as well as assigning of atomic radii and charges were performed automatically within the server. MEAD numerically solves the Poisson-Boltzmann equation to yield the distribution of electrostatic potential on the protein surface. Calculations were performed on one of the native a-chains of the 2HbS crystal structure (Harrington et al, 1997) as well as its SCWRL (Dunbrack et al, 1993) generated mutants. All calculations were performed by setting the internal protein dielectric constant to 4 and the external solvent dielectric constant to 80. The ionic strength parameter was held at 0.1

The time kinetics of deoxyhemoglobin polymerization were studied in 1.8M, 1.5 and 1 M potassium phosphate buffer (pH 7.25) respectively as described by Adachi and Asakura (1979a, b) using a Cary 400 spectrophotometer equipped with a Peltier temperature controller. Deoxygenation of the hemoglobin sample was ensured by passing moist gaseous nitrogen over the sample in an airtight cuvette and by addition of sodium dithionite. The polymerization of the resultant deoxyhemoglobin samples was initiated by a temperature jump from 4 to 30 oc within 10 sec and the progress of the reaction was followed by monitoring turbidity changes at 700 nm. The delay time was calculated from the kinetic traces

with the plunger of a Hamilton syringe. The tube was centrifuged at room temperature at 14,000 rpm for 30 min. The above process of gel-disruption and centrifugation was repeated twice subsequent to which the oil layer was aspirated and suitable aliquots from the supernatant were taken for estimation of Csat by Drabkin' s reagent (Goldberg eta!, 1977).

he gelation concentrations of HbS constructs were determined by the dextran-Csat method of Bookchin et a! (Bookchin et a!, 1999). This method allows measurement of Csat under near-physiological conditions and at much lower concentration of HbS (about 5-fold or less) than that required in standard Csat assays, but essentially provides the same information. Briefly, a suitable aliquot of a concentrated solution of hemoglobin in potassium phosphate buffer (0.05 M, pH 7.50) was taken in a 1.5 ml micro-centrifuge tube. A concentrated dextran (70 kDa) solution prepared in the same buffer was added to it and mixed well. This mixture was overlaid with 0.5 ml of mineral oil, chilled on ice bath and deoxygenated with an anaerobically prepared dithionite solution through an airtight Hamilton syringe. The final concentrations of dextran and dithionite in the mixture were 120 mg/ml and 0.05 M respectively. The deoxygenated sample above was allowed to polymerize at 37°C for 30 min after which the gel under the oil layer was disrupted

All experiments were carried out on a Beckman XL-A analytical ultracentrifuge, equipped with absorbance optics, and an An60-Ti rotor, at 20 °C. Sedimentation velocity experiments were performed at 40,000 rpm. Data were collected at 540 nm and at a spacing of 0.005 em with three averages in a continuous scan mode. The protein concentration varied in the range 4-40 IJ.M (heme) in 50 mM phosphate buffer, pH 7.2

acid. The respective buffer baselines were subtracted from the sample CD data. The ellipticity of the protein samples is reported as mean residue ellipticity (MRE) in deg/cm2/dmol units. The first derivative UV spectra of the oxy and deoxy-HbS were recorded on a Lambda Bio20 spectrophotometer (Perkin Elmer Life ScieAces). The hemoglobin concentration used for the spectral measurements was approximately 50 )!M on heme basis.The spectra of unliganded proteins was recorded subsequent to deoxygenating the hemoglobin samples by passing moist gaseous nitrogen extensively over the sample in an airtight cuvette. Completion of deoxygenation was ascertained by recording the visible spectrum of the deoxygenated Hb sample.

Circular dichroism (CD) spectra were recorded on a J71 0 Spectropolarimeter (Jasco, Japan) fitted with a Peltier type constant temperature cell holder (PTC-348W). The calibration of the equipment was done with (+)-! 0-camphorsulfonic

The synthetic peptides were purified by RPHPLC on an Aquapore RP300 column (250 x 7 mm) using a 4-72% linear gradient of solvent B (acetonitrile containing 0.1% TFA) in 130 min at a flow rate of 2 mllmin. Globin chains from respective hemoglobins were separated on a similar column of a smaller dimension (250mm x 4.6 mm) under identical conditions but at a flow rate of0.7 ml/min. Electro spray mass spectrometric analysis was carried out on a VG Platform (Fisons) mass spectrometer. The instrument was usually calibrated with standard horse heart myoglobin or gramicidin S solution. Appropriate amount of each sample was taken in 50% acetonitrile containing I% formic acid and analyzed under the positive ion mode.

Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.

concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column

Construction of mutant a globins

The semisynthetic a globin was purified from the mixture by CM52 urea chromatography as explained below. The lyophilized sample was dissolved in 0.005 M phosphate buffer (pH 6.9) containing 8 M urea and 0.05 M 2-mercaptoethanol at a concentration of I 0-15 mg/ml and loaded onto a CM52 column (16 em x 1.5 em) equilibrated with the same buffer. After an initial wash with the same buffer, two linear gradients of(a) 100 ml each of0.005 M to 0.03 M and (b) 100 ml each of0.02 M to 0.05 M phosphate buffer (all buffers contained 8 M urea and 0.05 M 2-mercaptoethanol, and were adjusted to pH 6.9) were employed at a flow rate of 45 ml/h to elute the semisynthetic a globin. The column was finally washed with 0.05 M buffer to elute unreacted a31-141 fragment from the column. The elution profile was monitored at 280 nm. The fractions for semi-synthetic a globin were pooled, extensively dialyzed against 0.1% TF A and lyophilized. The semisynthetic yield of the protein varied between 35% to 45%

V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized

Peptides were synthesized by standard solid phase synthesis protocols using Fmoc chemistry on a semi-automated peptide synthesizer (Model 90, Advanced Chemtech). For this, Wang resin pre-loaded with N-a-Fmoc-Glu was used as the starting material. The stepwise coupling of Fmoc amino acids was performed with DIPCDIIHOBT activation procedure. The coupling of each step was monitored by Kaiser test for free amine and wherever necessary, a double coupling was used to increase the yield. Before each coupling step and on completion of the synthesis, the N-terminal Fmoc group was removed using 20% piperidine (v/v in DMF). The peptides were cleaved from the resin and the side chains deprotected with appropriate volume of a mixture containing TF A, ethanedithiol, phenol, thioanisole and water (80:5:5:5:5, v/v). The resin was removed by filtration and the crude cleaved peptides were precipitated using cold diethyl ether and extracted in water. The peptides were purified by RPHPLC and their chemical identity was checked by mass spectrometry

The complementary segments of a globin needed for the semisynthesis of mutant chains were prepared by V8 protease digestion (Sivaram eta/, 2001). The a globin was dissolved in 0.01 M ammonium acetate buffer (pH 4) at a concentration of 1.0 mg/ml and digested at 37°C with V8 protease (1: 200, w/w) for 3 hours. The completion of digestion was ascertained by RPHPLC, after which the reaction was quenched by addition of neat TFA to a final concentration of 0.1 %. The complementary segments, al-30 and a31-141, from the digestion mixture were isolated in pure form by size-exclusion chromatography on a Sephadex G50 column (98cm x 2.8cm). The column was equilibrated and run in 0.1% TFA. The lyophilized sample of the digest was dissolved in the above solvent and loaded on to the column. The column was run at a flow rate of 30 mllhour and the elution profile monitored at 280 nm. The individual chromatographic profile of a globin digest showed only two peaks, a31-141 and a1-30 respectively, as expected from a single cleavage at the 30-31 peptide bond. The peak fractions were pooled separately and lyophilized

The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded

The [3-PMB and a-PMB chains were eluted with a linear gradient of 500 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) at a flow rate of 50 ml/hour. The chains were separately concentrated using Centriprep concentrators (Amicon) and stored in liquid nitrogen till further use

The heme bound a and ~ subunits were obtained as described by Bucci (1981 ). Briefly, hemoglobin was reacted with PMB in an eight fold molar excess (8 moles of PMB per mole of hemoglobin). The reaction mixture was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and then loaded onto a CM52 column (30cm x !Scm) that was pre-equilibrated with the same buffer.

Blood was drawn from appropriate source into heparinised tubes. The blood sample was centrifuged at 4000 rpm for 15 min ( 4 °C). The supernatant was discarded, and the erythrocytes (pellet) were subsequently washed thrice with chilled isotonic buffer [0.01 M PBS (pH 7.4)] by centrifugation at 4000 rpm and 4°C for 15 min. The washed erythrocytes were lysed in water. The resultant red cell lysate was then dialyzed extensively against PBS (pH 7.4) at 4°C to obtain stripped hemoglobin (hemoglobin devoid of bound allosteric modulators like BPG). The stripped hemoglobin was then loaded onto a pre-equilibrated DE52 column (30cm x 15cm) after extensive dialysis against 0.05 M tris acetate buffer (pH 8.5). The protein was eluted from the column employing a linear gradient of 500 ml each of 0.05 M tris acetate (pH 8.5) and 0.05 M tris acetate (pH 7) at a flow rate of 50 ml/hour. The purified hemoglobin was estimated spectrophotometrically at 540 nm (molar extinction coefficient= 53236 cm-1/M) and stored at -70°C till further use.

Fmoc protected amino acids and other chemicals used in peptide synthesis were obtained from Novabiochem (Switzerland). V8 protease and TF A were procured from Pierce Chemical Company (USA), while }-propanol, PMB," Hemin, Dithiothreitol, EDT A were obtained from Sigma Chemical Company (USA). DE52 and CM52 ion exchange resins were purchased from Whatman (UK). Sodium diothinite was procured from Fluka (Switzerland) and Catalase from Boehringer Mannheim (Germany). Carboxypeptidase was obtained from Worthington Biochemical Corporation (USA).

Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads

The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field

Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.

For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m

For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning

and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.

4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS

1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF

20 mg/ml X-gal in dimethylformamide Solution A as 40 mM potassium ferricyanide. Solution B as 40 mM potassium ferrocyanide. Solution C as 200mM magnesium chloride. 10X fixative (20% formaldehyde; 2% glutaraldehyde in 10X PBS) 10X PBS as 0.017 M KH2PO4, 0.05 M Na2HPO4, 1.5 M NaCl, pH 7

This protocol is for the detection of β-gal expression in fixed cells. It was performed on 96-well plates for initial screening of tTA transfected clone, and is a modification of Sanes et al., 1986

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice

Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were

After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA

The PCR parameters were as follows

The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).

appropriate secondary antibody (conjugated with horse-radish peroxidase) diluted in 5% fat free milk solution (in PBST) and incubated for 45 minutes at room temperature. After incubation the membrane was washed and processed for the detection of protein bands using ECL-plus detection reagent (Amersham Biosciences) followed by detection of signal on X-ray film (Hyperfilm-ECL, Amersham Biosciences)

The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with

Immunobloting

For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel

Lysate Preparation for Immuno- blotting

DTT. Then contents were then mixed and 1μl (200 units) of M-MLV was added. The mixture was then incubated at 37°C for 50 minutes. The reaction was stopped by incubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then used as a template for PCR. The expression of the investigated genes was determined by normalizing their expression against the expression of actin or GAPDH gene

Semi-quantitative RT-PCR

μg of total RNA was reverse-transcribed using poly-T oligonucleotide and M-MLV reverse transcriptase (Invitrogen) according to manufacturer’s protocol. Briefly, a 20μl reaction volume was made for 1μg of RNA. In a microcentrifuge tube, 1μl oligo (dT)(500μg/ml) , 1μg total RNA, 1μl 10mM dNTP mix and sterile water was added to afinal volume of 13μl. The mixture was then incubated at 65°C for 5 minutes ad quickly chilled on ice. To this mixture were added 4μl of 5X first strand buffer and 2μl of 0.1M

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed, from 35mm dish and 1ml to TRIzol was added directly to the dish and kept at room temperature for 5 minutes. The cells were harvested by pipetting up and down three four times and transferred to a 1.5ml microfuge tube. For each 1mlTRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed for 10 minutes. The upper aqueous phase was transferred into a fresh microcentrifuge tube and 500 μl of isopropanol was added and this was incubated at -20°C for 1 hour. The RNA was pelleted by centrifugation at maximum speed for 30 minutes at 4°C. The supernatant was decanted and the pellet washed with 1ml of 70% ethanol followed by a second wash with 1ml of 90% ethanol and centrifugation at maximum speed for 10 minutes. The supernatant was removed and the pellet air-dried for about 5 minutes and re-solubilized in 30-50 μl RNase free deionized (DEPC-treated Milli-Q) water and aliquots were stored at -70°C

Transient transfection of plasmid DNA in culture cells was performed using Lipofectamine 2000 according to manufacturer’s protocol. Briefly, forty million cells were seeded in a 35mm tissue culture dish, one day before transfection. Transfection was performed 18-24 hrs after seeding the cells. 4μg DNA was mixed in 50μl of Opti-MEM in one eppendorf tube. In another tube, 5μl of Lipofectamine 2000 was diluted in 50μl Opti-MEM and incubated at room temperature for 5 minutes. After five minutes, DNA and Lipofectamine 2000 were mixed together and complexes, incubated for 30 minutes at room temperature. Meanwhile, the adherent cells were washed twice with PBS and 1ml of Opti-MEM was added. 100μl of complexes were then added to each dish containing cells and medium. After 6hrs, the medium containing complexes was removed and complete medium was added and transgene expression was accessed 24-48 hrs after transfection

Transient transfections in adherent cells

The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by taking 1 OD 260= 50 μg/ml for DNA, 40 μg/ml for RNA and 33 μg/ml for single stranded oligonucleotides. The purity of nucleic acids was checked by their A260/A280 ratio

ethanol has dried. The pellet was resuspended in 20 μl of milliQ water and 20 μg/ml RNase added. The tube was incubated at 50°C for 45 min. the tube was vortexed for few seconds. Quality of the plasmid DNA was then accessed by running 1% agarose gel.

stored. To prepare competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate, inoculated into 3 ml LB medium, and incubated overnight at 37°C temperature with shaking at 200 rpm. 1% of this pre-inoculums was sub cultured in 100 ml LB-broth and incubated at 18°C with shaking until OD at 600nm reached 0.5 - 0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking.Cells were pelleted by centrifugation at 2000 g at 4°C for 8 min. Pellet was resuspended in 40 ml of ice-cold TB buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 g at 4°C for 8 min. Pellet was resuspended in 8 ml of TB buffer in which final concentration of DMSO was 7% and left on ice for 10 min. 100 μl aliquots were made and snap frozen in liquid nitrogen and stored at -80°C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.2H2O) except MnCl2 were dissolved in milliQ water and pH was adjusted to 6.7 with 1N KOH. MnCl2 was dissolved separately in mill Q water. MnCl2 was added drop wise while stirring (MnCl2 if added directly will give a brown color to the solution and precipitate out, hence it needs to be dissolved separately). Solution was then filter sterilized and

Overnight Grown culture was pelleted by centrifugation at 10,000g at 4°C for 3 min and the supernatant was discarded. Pellet was resuspended in 250 μl of ice-cold alkaline lysis solution 1. 300 μl of alkaline solution 2 was then added and the tube was inverted gently 3-4 times and incubated at room temperature for 5 min. 350 μl of ice cold solution 3 was added and mixed by inverting the tube rapidly for 3 or 5 times. Suspension was incubated on ice for 10 min. Bacterial lysate was spun at 10,000g for 12 min at 4°C. Supernatant was transferred to a fresh tube. 0.4 volume of phenol: chloroform was added to the supernatant and the contents mixed. It was then spun at 10,000g at 4°C for 12 min. Aqueous phase was taken out in a fresh tube and 0.6 volume of isopropanol was added, mixed properly and incubated at room temperature for half an hour followed by spinning at 10,000g at RT for 20 min. Supernatant was discarded. Pellet was washed with 70% ethanol. The tube was stored at room temperature until the

250 mM KCl 55 mM MnCl2.4H20 50 x TAE (1 litre): 242 g of tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA Alkaline Lysis Solution 1: 50mM tris-HCl (pH 8.0) 10.0 mM EDTA 50 mM glucose Alkaline Lysis Solution 2: 0.2M NaOH 1% SDS Alkaline Lysis Solution 3: 3.0M Potassium acetate

2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4Tris Buffer Saline (TBS) (10X): 12.1gm Trizma Base 40.0gm NaCl Adjust PH to 7.6; make up the volume to 1 lit with milli Q water. HEPES Buffer Saline: 20 mM HEPES (pH 7.5) 150 mM NaCl Blocking Buffer: 5% fat free milk or 2% BSA in PBST or TBST. Stripping Buffer: 100 mM β-mercaptoethanol 2% (w/v) SDS 62.5 mM Tris-HCl (pH6.7) Luria Broth: 10g tryptone 10g NaCl 5g yeast extract, make up the volume to 1 lit with water. TB buffer for preparation of competent cells: 10 mM PIPES (free acid) 15 mM CaCl2.2H20

2.5 ml of 1.5 M Tris-Cl (pH 8.8) 4.0 ml of 30% acrylamide; bisacrylamide (29:1) mix 50.0 μl of 20% SDS 3.35 ml of milli-Q water 100 μl of 10% APS 10.0 μl of TEMED. 2X SDS loading Buffer: 130 mM Tris-Cl (pH 8.0) 20% (v/v) glycerol 4.6% (w/v) SDS 0.02% bromophenol blue 2% DTT SDS PAGE Running Buffer: 25mM Tris base, 0.2M glycine 1% SDS Western Blot: 1 x Blotting Buffer (2Litres): 25mM tris base, 0.2M glycine 20% methanol Phosphate Buffer saline (PBS): 137 mM NaCl

Whole cell lysis buffer: 20mM Tris (PH 7.5) 150mM NaCl 1mM EDTA 1mM EGTA 1 % triton X 100 2.5mM sodium pyrophosphate 1mM β-glycerophosphate 1mM Na3VO41μg/ml aprotinin, 1μg/ml leupeptin and 1μ.ml pepstatin SDS-PAGE: Stacking Gel Mix (4ml, 5%): 380μl of 1M Tris-Cl (pH 6.8) 500μl of 30% acrylamide ; bisacrylamide (29:1) Mix 15 μl of 20% SDS 2.1 ml of milli-Q water 30 μl of 10% APS 5 μl of TEMED. 12% Resolving Gel Mix (10ml):

Human lung epithelial type II cells (HPLD) were a kind gift from Dr. T Takahashi, Japan. All the cell lines were maintained in 5% CO2 with the recommended media containing 10% FBS (3% FBS for HPLD) and following the standard guidelines.

A549 (human lung adenocarcinoma epithelial) and murine fibroblast cell line NIH3T3 was purchased from American Type Culture Collection. Murine lung epithelial cell line E-9 and E-10 were a kind gift from Dr. L.M.Anderson, (NCI-FCRDC, Frederick, Maryland)

Monoclonal antibody against KRAS were purchased from Merck Research Laboratories, phospho p44/42 (ERK1/2)and total p44/42 (ERK1/2)antibodies were purchased from Cell Signaling Technologies. Anti tubulin antibody was obtained Sgima-Aldrich Chemicals. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bangalore Genei Pvt. Ltd.

All the chemicals used for routine molecular biology work were procured from Sigma-Aldrich Chemicals (St Louis, MO, USA) unless otherwise mentioned. Taq polymerase for PCR and standard DNA markers and protein markers were purchased from MBI Fermentas. Tissue Culture materials like DMEM medium (for A549), Ham’s F-12 medium (for HPLD), Opti-MEM medium, 0.5% trypsin-EDTA, 100X antibiotic-antimycotic, freezing medium, fungizone, 200mM L-glutamine, fetal bovine serum (FBS), Lipofectamin-2000 and TRIzol were obtained from GIBCO BRL (Gaithersburg, Maryland, USA). CMRL medium was purchased from ICN laboratories. M-MLV reverse transcriptase, RNase inhibitor, dNTPs and MgCl2 were obtained from Invitrogen Corporation (Carlsbad, CA). ECL western detection kit and HybondTM- P were purchased from Amersham biosciences (GE Healthcare, UK).

f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine

a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.

d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine

One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once

b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine

5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once

5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine

Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA

Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised

12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml

A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml

1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl

microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots

The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml

These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)

Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)

the membrane and sandwiched between 1 piece of buffer-soaked Whatman paper from Biorad on each side. The sandwich was placed between graphite electrodes with the membrane towards the anode. The transfer was done for 12-16 hr using a voltage of 40 V in the cold room. Protein transfer was viwed using Ponceau S staining. Blot was dipped in the Ponceau S stain under shaking and washed using PBST. After transfer, the membrane was blocked with 5% non-fat milk in PBST (1X PBS with 0.1% Tween-20) for 2 hr at room temperature. The membrane was then washed thrice with PBST under shaking and incubated with the primary antibody (1:1000 dilution in PBST) for 12-16 hr. The membrane was again washed thrice under shaking with PBST and incubated with 1:20000 dilution alkaline phosphatase conjugated anti-goat IgG secondary antibody (in PBST) for 2 hr. The membrane was once again washed thrice as described above and the signal developed using ECL kit from Amersham on X-ray films in a dark room. The reactive protein bands appeared as black bands upon gentle shaking at room temperature in 1X developer solution. The reaction was stopped by dipping and shaking the film in 1X Fixer solution followed by washing in water

The protein samples separated on SDS-PAGE were transferred to PVDF (polyvinyledene difluoride) membrane (Amersham, Buckinghamshire, UK) electrophoretically by a semi-dry method using BioRad apparatus. The gel and the membrane (pre-wetted with methanol) were wetted with transfer and the gel was placed in contact with

(ii) Stacking gel buffer: 1.0 M Tris-Cl pH 6.8 (iii) Resolving gel buffer: 1.5 M Tris-Cl pH 8.8 (iv) SDS stock: 10% (w/v) solution (v) Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh (vi) Gel running buffer (1X) (vii) Loading dye (6X): (viii) Lysis buffer (RIPA) Gels of 1.5 mm thickness were cast in the Biorad small gel apparatus. Resolving gel of 10% (10 ml) was made by mixing 4.2 ml 10% acrylamide, 3.1 ml water, 2.5 ml of 1.5 M Tris-Cl pH 8.8 and 0.1 ml of 10% SDS. Stacking gel (2 ml) was made by mixing 0.33 ml of 30% acrylamide, 1.4 ml of water, 0.25 ml of 1 M Tris-Cl pH 6.8 and 0.02 ml of 10% SDS. Gels were polymerized by the addition of TEMED (N,N,N′, N′-tetramethyl ethylene diamine) and APS (1/100th volume of gel mix). Sample preparation for gel loading was done as follows. Mid log and late log phase 10 ml cultures were centrifuged at 26000g and the cell pellet was resuspended in 0.5 ml RIPA buffer. Cells were sonicated on ice for 1 min at output power of 5 to get a cleared lysate. The culture lysate was centrifuged at 26000g to recover the clear supernatant. Total cell protein was quantified in the lysates using BCA kit reagents (BioRad) using the manufacturers protocol. Appropriate volume of cell lysate was mixed with the loading dye in a final concentration of 1X and loaded onto the gel. The gel was run at constant voltage of 60 V for stacking and 80 V for resolving gel

The method followed was as described in Sambrook and Russell (2001). The following solutions were used to cast and run SDS-PAGE gels. (i) Acrylamide stock: 29% (w/v) acrylamide and 1% N,N′-methylene bisacrylamide

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions by either CDFD or an outsourced sequencing facility

Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

Denaturation 95°C 1 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

The PCRs were normally performed using a PCR amplification kit from Fermentas/Sigma (USA), following the company's protocols. Approximately, 10 ng of chromosomal or 1-2 ng of plasmid DNA was used as template in a 50 μl reaction volume containing 0.2 mMM of each dNTP, 20 picomoles each of forward and reverse primer and 0.5 units of Taq DNA polymerase. In some cases, freshly streaked E.coli cells from a plate were resuspended in 50 μl of sterile Milli Q water to get a cell suspension (~ 109 cells/ml) and 10 μl from this was used as the source of DNA template. The samples were subjected to 30 cycles of amplification and the typical conditions of PCR were as follows (although there were slight modifications from one set of template/primers to another): The initial denaturation was done at 95°C for 3 min and the cycle conditions were as given below. Annealing 55°C 1 min Elongation 72°C (1 min/kb of DNA template to be amplified)

Fresh overnight culture of the E. coli strain (DH5α) was subcultured 1:100 in 250 ml LB/SOB media at 18 ̊C and 2500g and allowed to grow to an A600=0.55. Culture was chilled on ice and centrifuged at 2500g at 4 ̊C for 10 min. The cell pellet was redissolved in 80 ml ice-cold Inoue transformation buffer (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES pH6.7). This cell suspension was centrifuged at 2500g at 4 ̊C for 10 min. and the cell pellet was resuspend in 20ml ice-cold Inoue buffer with 1.5 ml DMSO. This mixture was then placed on ice for 10 min. Aliquots of this suspension were dispensed into chilled, sterile microfuge tubes that were snap-frozen in a bath of liquid nitrogen. Tubes were stored at ─70 ̊C until required. For transformation the cells were thawed on ice and plasmid DNA was added followed by the standard transformation protocol

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Typically, 100-200 ng of DNA was used in each ligation reaction. The ratio of vector to insert was maintained between 1:3 and 1:5. The reactions were usually done in a 10 μl volume containing ligation buffer (provided by the manufacturer) and 0.05 Weiss units of T4 DNA ligase, at 16°C for 12-16 hr

0.5-1 μg of DNA was used for each restriction enzyme digestion. 2-4 units of the restriction enzymes with the appropriate 10X buffers supplied by the manufacturers were used in a total reaction volume of 20 μl. The digestion was allowed to proceed for 6 h or 10min. (for FAST digest enzymes) at the temperature recommended by the manufacturer. The DNA fragments were visualized by ethidium bromide staining following electrophoresis on 1-1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

The DNA samples were mixed with the appropriate volumes of the 6X loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 1-1.5% agarose gel in either 1X TBE or 1X TAE buffer. The gel was stained in 1 μg/ml of ethidium bromide solution for 30 min at room temperature and the bands were visualized by fluorescence under UV-light

Quiagen/HiPura following the manufacturer's protocols

The rapid alkaline lysis method of plasmid isolation, as described by Sambrook and Russel (2001), was followed with minor modifications. Bacterial pellet from 3 ml of stationary-phase culture was resuspended in 200 μl of ice-cold solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0 containing 1 mg/ml lysozyme) by vortexing. After 5 min incubation at room temperature, 400 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed, by gently inverting the tube several times. This was followed by the addition of 300 μl of ice-cold solution III (5 M potassium acetate, pH 4.8) and gentle mixing. The tube was incubated on ice for 5 min and centrifuged at 20,0000g for 15 min at 4°C. The clear supernatant was removed into a fresh tube and, if required, was extracted with an equal volume of phenol:chloroform mixture. The supernatant was precipitated with either two volumes of cold 95% ethanol or 0.6 volumes of isopropanol at room temperature for 30 min. The nucleic acids were pelleted by centrifugation, washed with 70% ethanol, vacuum dried, and dissolved in appropriate volume of TE buffer. If required, the sample was treated for 30 min with DNase free RNase at a final concentration of 20 μg/ml. The plasmid DNA was checked on a 0.8% agarose gel and stored at −20°. The plasmid DNA thus isolated was suitable for procedures such as restriction digestion, ligation, and preparation of radiolabeled probes. Plasmid isolation was also done with any of the commercially available kits from

and the aqueous phase transferred to a fresh tube. The aqueous phase was further extracted successively, first with phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform:isoamyl alcohol (24:1). DNA was precipitated from the clear supernatant by the addition of 0.6 volumes of isopropanol. The chromosomal DNA was either spooled out or pelleted at this stage, washed with 70% ethanol, air-dried, and dissolved in suitable volume of TE buffer

The method as described in the manual Current Protocols in Molecular Biology was followed for preparation of chromosomal DNA. Cells from 1.5 ml stationary phase culture were recovered by centrifugation and resuspended in 567 μl of TE buffer. To this, 30 μl of 10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cell suspension mixed and incubated at 37°C for 1 h. Next, when the suspension looked cleared, 100 μl of 5 M NaCl was added, thoroughly mixed, followed by the addition of 80 μl of CTAB/NaCl (10% cetyltrimethylammonium bromide in 7 M NaCl) and vigorous mixing (by inverting the microfuge tube). The suspension was incubated at 65°C for 10 min, brought to room temperature, extracted with an equal volume of chloroform-isoamyl alcohol (24:1 v/v)

Extraction of chromosomal DNA from bacterial cells

The method followed is essentially the same as described by Jin et al. (1992). Overnight bacterial cultures grown in minimal A medium supplemented with 0.4% glycerol and 0.5% Casamino acids with the appropriate antibiotic were subcultured 1:100 in the same medium in a volume of 20 ml (0.2% arabinose was added for induction of the plasmid-borne gene downstream of Para, wherever required) at 37 ̊C. Cultures were induced with 1 mM IPTG at A600=0.3. 1 ml samples were aliquoted at time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 1 ml of 0.1 mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling the cultures were incubated at 37 ̊C for 15 min. 0.5 ml of this culture was then taken in duplicate tubes for β-galactosidase assays

high osmolarity conditions (Gowrishankar, 1989; Csonka, 1989) for β-galactosidase assay

Assays for determination of β-galactosidase enzyme activity in cultures were performed as described by Miller (1992) after permeabilizing the cells with SDS/chloroform, and the activity values were calculated in Miller units, as defined therein. For determination of proU activity from a proU::lac fusion that contains the proUpromoter cloned upstream of the lacZYA genes (as in plasmid pHYD272), cultures used were grown in LBON or K-medium (low osmolarity medium) since proU is also induced under

β-Galactosidase assay

a very sensitive test and is often used for determining the viability of a strain in the presence or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and the appropriate antibiotic and plating an appropriate dilution (10─5 and 3X10─6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. The ratio of the number of colonies obtained on the –IPTG plate to that on the +IPTG plate determined the efficiency of plating. The colonies from the test plate (that is, ─IPTG plate) were also subsequently subcultured on the same medium (─IPTG) to determine viability of the strain. Likewise, strains carrying Ts plasmids were cultured overnight at 30 ̊C with the appropriate antibiotic and dilutions of this culture (10─5 and 3X10─6) were plated at two temperatures 30 ̊C (permissive) and 37 ̊C or 39 ̊C (non-permissive or test). The ratio of the number of colonies obtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature. Viability of the strain was subsequently confirmed after subculturing from the test plate as stated above

Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is

Strains were grown overnight in LB containing 0.4% maltose and 10 mM MgSO4,subcultured and grown to early stationary phase in the same medium. 100 μl of the culture was mixed with 2.5 ml of soft agar and overlaid on LB agar plates supplemented with 0.4% maltose and 10 mM MgSO4. Serial dilutions of λcI857 lysate were prepared (in LB) and 10 μl were spotted from each dilution (and the undiluted) on the soft agar lawn and allowed to dry. The plates were incubated at the appropriate temperature overnight, and the plating efficiency determined

The lacZ U118 is an amber nonsense mutation(Am) that confers Lac─phenotype and also polarity of the downstream lacYA genes in the operon due to premature Rho-dependent transcription termination within the untranslated region of lacZ. Melibiose is a sugar which can only be utilized in a lacZ (Am) strain at high temperature (39 ̊C, when the native melibiose permease is inactive) if the downstream gene lacY encoded permease is transcribed and translated. Therefore, in lacZ (Am) strains, growth on minimal melibiose plates (0.2%) at 39°C reflects transcriptional polarity relief at the lac locus, and the same was scored after streaking the relevant strains on such medium

dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium

The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-