due 24 hours
jacckkyy
lab_result_introq.pdf
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MMG 408. EXPERIMENTS I - III Experiment I: Bacterial Growth and Induction of Bacterial Operons, Bacterial Genetics Exponential Growth and Induction of the E. coli lac Operon All types of bacteria multiply by binary fission so their growth is exponential. As a consequence, if the number of bacteria in a growing bacterial culture is plotted versus the time of growth on semilog graph paper, a straight line will result. The optical density (OD) of the culture is due to light scattering by the bacteria in the culture and is proportional to the number of bacteria in the culture. To conserve energy, cells often only express genes that are required for maximum growth under the conditions the cells are in. For example, they will only express genes whose products are required to use a particular carbon and energy source if that carbon and energy source is present in the medium. Then, and only then, will they induce the transcription of the genes to use that carbon source. The genes of the lac operon are the classical example of inducible genes. This operon consists of a gene whose product transports lactose into the cell (lacY) and a gene whose product degrades lactose to glucose and galactose (lacZ). The product of the lacZ gene is the enzyme, β-galactosidase, that can be easily assayed using the β-galactoside, ortho-nitrophnyl-galactoside (ONPG), or 5-bromo-4- chloro-3-indoyl-β-D-galactoside (X-Gal) which turn color when cleaved by β-galactosidase.
Note: This information is provided to you so that you may have a rudimentary overview of the experiment you are about to perform. You may reference this information in your reports but
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must cite this work (even if you reference it in your methods). You may not simply rephrase these background sentences as that would constitute plagiarism just as simple copying would.
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Experiment I: Day 1 (9/1) Using Escherichia coli Mutants to Understand the lac Operon 1. Take two minimal, Histidine, Tryptophan, Arginine, X-Gal plates and divide them into thirds with a marker. 2. Using your spread plating technique, spread 100µL of the inducer (100mM IPTG) on one plate. 3. Streak (for isolation) each of the three mutants; E. coli CB2881 (lac+), E. coli PK191 (lacZ-), and E. coli
CB846 (lacI-) in one third of each plate. Complete the accompanying worksheet for Experiment I: Day 1. It should be included in your report. Follow-Up 1. Record your observations. Did each mutant behave as expected? Why or why not? Can you continue using the E. coli CB2881?
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Experiment I: Day 1 Worksheet. Name: To be completed during the lab period. This and all worksheets must be stapled with your report.
1. What is x-gal in this experiment? What is the mechanism of color change?
2. Briefly explain the action of IPTG.
3. One of the Escherichia coli mutants is always producing β-galactosidase. What is the term for this type of expression?
4. Record your color predictions below.
-IPTG +IPTG
E. coli CB2881
(lac + )
E. coli CB846
(lacI - )
E. coli PK191 (lacZ
- )
E. coli CB2881
(lac + )
E. coli CB846
(lacI - )
E. coli PK191 (lacZ
- )
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Experiment I: Day 2 (9/6): Generating a growth curve for Escherichia coli CB2881 Working in pairs. An overnight culture of E. coli CB2881 has been diluted for you to an OD625nm of approximately 0.05 (likely 1:50).
1. Remove one 5ml tube from the 37°C shaker and label it with your name. 2. Blank a spectrophotometer with sterile LB at 625nm (Note: mark the top of your tubes and blank using
the same side each time). 3. Measure and record the optical density (O.D. 625nm) of your freshly diluted culture (Note: mark the top
of your tubes and read the same side each time). 4. Return the tube to the shaking incubator. 5. At 30 minutes intervals, record the O.D. 625nm of the culture. 6. At 90 minutes, and 120 minutes, remove a 20µl sample for viable cell counts. 7 To do this, place 180 µl sterile saline solutions into each of the 7 microfuge tubes. 8 Add 20 µl of the bacterial culture to the first, mix and transfer 20 µl to the next tube. 9 Continue this operation until the last tube. Remember to change tips at every dilution! 10 Using spread plate technique; plate the last 3 dilutions on LB agar (using 100 µl on each plate).
Preparation for Experiment II Selection of rifampin resistant mutants (rpoB) Note: This activity will be reported in the lab report for Experiment II. You will need to record your methods and data in your notebook as usual, but will not be reporting on them until Experiment II is completed.
1. Centrifuge 1ml of the overnight Escherichia coli HR171 (rifS) in a microfuge for 1 minute. 2. Decant the supernatant and resuspend the cells in the LB that remained on the walls of the tube after
decanting (around 100-200 µl only). 3. Spread the whole volume on an LB with rifampin plate. 4. Incubate at 30°C.
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Experiment I: Day 3 (9/8) Follow-up of determination of growth curve:
1. After 1-2 days of incubation, count the number of colonies on your plates. 2. Back-calculate the concentration of viable cells in the culture at each of the sampling points. 3. Plot the data as O.D. 625nm vs. time (minutes) and as colony forming units/ml versus time on the semi-log
graph paper.
Preparation of buffer for β-galactosidase assay Each student calculates how to prepare 100 ml solution of a buffer of the following composition:
Na2HPO4 H2O 60 mM NaH2PO4 H2O 40 mM KCl 10 mM MgSO4 1 mM di-thio-Threitol 30 mM CETAB 0.15% pH 7.0.
Check the calculation before preparing the buffer among your colleagues and let the instructor approve it. Prepare the buffer in groups one per bench. Note: Since weighing small quantities has a considerable error we will provide you with 0.2 M solutions of the ingredients.
Measuring the Induction of the Escherichia coli lac Operon Your instructor has already diluted an overnight culture of E. coli CB2881.
1. Take 5-6 ml from the shaking incubator (250 rpm at 30°C) and label it with your name. 2. Check the OD625 nm using the spectrophotometer. Blank with LB. 3. Continue incubating the culture until it reaches an OD625 nm of 0.3 (takes about 2 hrs to reach O.D. of
0.3). 4. During this period prepare the assay by adding 0.5 ml (i.e. 500µl) of assay buffer (prepared fresh) to
four 13x100mm tubes. 5. Number the tubes 1-4. Keep tubes on ice. 6. When the culture reaches the target OD (0.3), add 0.5 ml to Tube 1. Mix well, leave on ice. 7. Add 50-52µl of IPTG to the remaining cells and continue shaking (Note: Don’t confuse IPTG (inducer)
with ONPG (substrate).. 8. After adding the inducer record the OD625 nm at 15, 30, and 60 minutes, and add 0.5 ml of each time
sample to Tubes 2 (i.e. 15 min.), 3 (30 min.) and 4 (60 min.), respectively, kept on ice. 9. Begin the β-galactosidase assay. 10. Vortex each tube for at least 10 seconds to permeabilize the cells. 11. Warm the tubes for a few minutes in the 30°C water bath. 12. Add 200µl of ONPG to each tube and return the tubes to 30°C water bath. (Note: It helps to stagger the
additions so that you have time to manipulate each tube. For example: make additions of ONPG at 30 seconds intervals).
13. Start timing the reaction when you add ONPG and place the tubes at 30°C.
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14. When tube #4 turns yellow (usually takes 15’), record the actual reaction time. 15. Begin stopping the reactions by adding 1.5 ml of 1M Na2CO3 (at the same 30 second intervals). Note:
Do not keep Na2CO3 on ice or it will come out of solution. 16. Using the spectrophotometer record both the OD420 nm and the OD550 nm. Blank with Assay buffer &
LB (1:1). The first of these measurements determines the intensity of yellow color due to conversion o ONPG to nitro-phenol, the second determines the turbidity of the mixture due to the bacterial cell suspension.
17. Calculate the specific activity for each tube. Continued Preparation for Experiment II Selection of rifampin resistant mutants (rpoB) Note: This activity will be reported in the lab report for Experiment II. You will need to record your methods in your notebook as usual, but will not be reporting on them until Experiment II is completed.
1. Count the number of colonies grown on your rif plate. 2. Streak out two rifR mutants on LB rifampin plates to purify [these came from plating E. coli strain
HR171 rifS on rifampin plates (see experiment I, day2), looking for spontaneous mutants). 3. Incubate at 30°C overnight.
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Questions to answer in your final report on Experiment I
1. What is the generation time of your bacterial culture?
2. How many viable bacteria per ml would be in your culture when the OD625 nm reaches 1.0? Plot growth both in OD625 nm vs. cells/ml and vs. time.
3. Why do the colonies of a wild type E. coli strain on the X-gal plate turn blue only when IPTG is also on the plates?
4. Why is the lacZ mutant never blue on X-gal? The lacI mutant always blue even without IPTG?
5. Why did it take some time after IPTG was added for the β-galactosidase in the cell to reach its maximum level
in units/mg protein?
6. What is the approximate specific activity (units per mg protein) of β-galactosidase in your culture each time before and after adding IPTG? Also express per cell. What would you have observed if you had done the induction with E. coli CB846? E. coli PK191?
€
Units mgprotein
=1000 × OD420 −1.75 × OD550 t × v × OD625 × 0.2
$
% &
'
( )
t = reaction time in minutes (the length of time you incubated the cells in ONPG) v = volume of cells you added per ml of reaction mix For example: if you added 0.2 ml cells to 2ml assay buffer, it would be 0.1. (Since you’ve added 0.5 ml cells to 0.5 ml assay buffer, then it should be 1.0)
OD625 nm = the OD the cells would have had at the time you removed them from the culture (OD ≈ 0.3?). We are assuming that 1ml of cells at OD625 nm = 0.5 has 100µg protein.
7. If the OD625 nm = 0.2 at the time IPTG was added, what would it be 1 hour later? Refer to your growth curve.
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Experiment II: Gene Mapping by Homologous Recombination In this experiment you will isolate a rifampin resistant mutant of Escerichia coli HR171. The rifR mutants occur spontaneously in a growing culture and will be selected on a rifampin agar plate. Once the desired mutant is isolated, you will map the mutation by conjugation and transduction. The E. coli chromosome is a circular DNA molecule about 4 x 106 base pairs in length. Normal E. coli is haploid; it contains only one copy of each of its genes. In order to map the order of genes on the E. coli chromosome, one measures the frequency of recombination among them using genes with differing alleles (i.e. a mutant allele like rifR and a wild type allele like rif
S ). Since the organism is haploid, for recombination to
occur one needs to transfer at least part of the chromosome of one strain (called the donor) into a cell of the other strain (the recipient). Experiment II uses two different methods to do this - conjugation and transduction. During conjugation DNA is passed from one bacterial cell to another by a conjugative plasmid. Normally, the plasmid only transfers itself but if it has integrated into the chromosome (an Hfr strain) it will also transfer the entire chromosome because the chromosome has become an integral part of the plasmid. We can use Hfr strains for mapping mutations because the farther a gene is on the DNA from the site of integration of the plasmid, the less frequently the gene will be transferred. Transduction is the transfer of DNA by a bacteriophage; bacterial DNA is packaged in a phage head and then enters another bacterium upon subsequent infection of this bacterium. We can use transduction for mapping because the phage head will only hold a small piece of bacterial DNA so for two genes to both be carried in the same head they must be close to each other in the DNA. The closer together they are, the more often they will be packaged together and cotransduced. Also, the frequency of the various recombinant types among the transductants will depend upon the order of the genetic markers.
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Genetic map of the Escherichia coli chromosome. The numbers refer to map position in minutes (minutes required for the gene to be transferred in conjugation), relative to the thr locus. 52 loci are shown, chosen on the basis of utility in mapping studies. Inside the circle, the leading transfer regions of a number of Hfr strains are indicated. The large arrow points at KL16, the transfer region of the Hfr strain employed in this experiment. The map positions of mutations of E. coli strains HR171 F- hisG argH trpA strR (rpsL), KL227 metA, and KL16-99 recA thi strS are shown with arrows. The region deleted in strain PK191 Δ(proB-lac)XIII is indicated by the dashed line.
strR
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Experiment II Transduction: Day 1 9/13 Preparing P1Cm c1-100 Lysogen
In the following order: 1. Place 100µl of an overnight culture of E. coli KL227 metA into a 13 x 15mm test tube. 2. Add 5 µl of 0.1 M CaCl2. 3. Add 50 µl of phage P1 c1-100 suspension provided. 4. Incubate at 32°C for 10 min to allow for phage adsorption and expression of CmR. 5. Plate 20 µl on LB – chloramphenicol (Cm 25 µg/ml) and incubate at 30°C. Note: Incubation at higher temperature will induce the phage to lyse the cells! Experiment II Conjugation: Start Cultures of Donor and Recipient 1. Inoculate 2ml of LB with one colony of Hfr KL16 (donor). 2. Inoculate 2ml of LB-rif with one colony of your mutant rpoB (recipient). Note: Try to choose a larger, faster growing colony. 2. Incubate in the 37°C shaker at 200rpm.
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Experiment II: Day 1 Worksheet. To be completed during the lab period. 1. What is a lysogen? 2. Why do the agar plates include chloramphenicol? 4. Why do we incubate the plates at 30°C and not 37°C?
Name:
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Experiment II Conjugation: Day 2 9/15) Hfr Cross
1. Dilute each culture (donor and recipient) 1:5 in fresh LB [(i.e. 1ml culture + 4ml LB, (2 tubes)]. 2. Label three 13x100mm tubes. 3. In tube 1 mix 500µL E. coli Hfr KL16 (donor) and 1ml of rpoB mutant (recipient). 4. In tube 2. dispense 500µL of E. coli Hfr KL16 5. In tube 3 dispense 1ml of rpoB mutant.
Tubes 2 and 3 are your controls of the donor and recipient, respectively. 6. Incubate without shaking for 90’ at 37°C in a water bath. 7. During incubation prepare M9 streptomycin plates by spreading 50µL of each amino acid as shown
below. Note: You must take care not to cross-contaminate the spreader. Rinse with the tap water squeeze bottle
between amino acids.
6. After incubation add the cultures to three separate microfuge tubes. 7. Centrifuge (max. speed) for no more than 30 seconds to pellet the cells. 8. Decant the supernatant carefully! 9. Resuspend the cells in 350µl of saline (repeat steps 7-9, in order to wash the cells from residual LB). 10. Spread the 100µl on each of the following plates:
11. Incubate plates at 37°C. Experiment II Transduction: Day 2 Continue propagation of cultures. 1. Streak out your rpoB mutant on a LB-Rif plate (from liquid culture). 2. Streak your P1Cm c1-100 lysogen on a fresh LB Cm plate and incubate at 30°C (from plate).
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Experiment II: Day 2 Worksheet. To be completed during the lab period. 1. Why do we incubate conjugation mix without shaking? 3. When you decant something, what are you doing?
Name:
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Experiment II Conjugation: Day 3 9/20 First Analysis of Transconjugants Work in groups of 4 so that each group has at least 40 transconjugants to analyze. 1. Examine the controls. 2. Assess the purity of all the plates. 3. Count the number of transconjugants on each of the selective plates. 4. Record your counts on the class spreadsheet. 5. Prepare 8 M9 streptomycin plates with trp and arg as before per group. 6. Purify 48 of the His+ conjugational recombinants.
Note: Pick isolated individual colonies only.
7. Save the plates with the P1 lysogen and of your rpoB mutant in and store them in the refrigerator for the next day.
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Experiment II Conjugation: Day 4 9/22 First Analysis of Transconjugants 1. Spread the appropriate amino acids to prepare the following plates: 2. Draw a line on the upper center of your petri dishes with a Sharpie. 3. Use the paper templates provided to place a small (3 – 4 mm) streak each of your colonies on each plate. 6. Do this using toothpicks or wires. Touch an isolated colony and patch onto each plate in this order: Experiment II Transduction: Day 4 Prepare Overnight Culture of P1 Lysogen 1. Inoculate a single, isolated colony of the lysogen into 5 ml LB. 2. Incubate at 30°C.
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Experiment II: Day 4 Worksheet. To be completed during the lab period. 1. What is the purpose of patching each of these plates? Tryptophan Only: Arginine Only: Rifampicin Only: Tryptophan and Arginine: 2. Why are we careful to patch onto the tryptophan and arginine plate last?
Name:
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Experiment II Transduction: Day 5 (9/27) Induction of the P1Cm c1-100 An overnight culture of the P1 lysogen of E. coli KL227 metA has been diluted for you. 1. Remove one tube from the shaker and label it as your own. 2. Check the OD625 nm. 3. Continue to incubate with shaking at 32°C. 4. Check the OD625 nm periodically until it reaches at least 0.6-0.7. 5. Transfer P1 lysogen culture to 42°C water bath shaker and shake vigorously for 30 min. 6. Transfer to 38°C and shake vigorously for 1.5 to 2 hours or until a lysis is detected.
Note: If there is time, you may proceed through steps 1-4 for Day 6. 7. Store phage stock at 4°C. Experiment II Conjugation: Day 5 Analysis of Transconjugants 1. Observe your patched plates. 2. Record the result from your Hfr cross and enter into a spreadsheet with class results. Follow-Up 3. Construct a “gradient of transfer graph” from your results and the pooled class results. Note: Use pages 206-207 of Snyder and Champness 2nd edition (pp. 179-180 of third edition) as a guide.
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Experiment II Transduction: Day 6 (9/29) Titration of Phage
8. Vortex your culture and check the OD625 nm to confirm the cells have lysed. 9. Add a drop of chloroform to your P1Cm c1-100 and vortex. 10. Transfer 1.5ml to a microcentrifuge tube. 11. Centrifuge for 2 minutes. 12. Transfer the supernatant to a fresh microcentrifuge tube. 13. Add another drop of chloroform and vortex. 14. Spin again for 1 minute. 15. Use 20 µl of the supernatant to serially dilute to 10-4, 10-5 and 10-6.
To do this:
16. Place 180 µl sterile saline solution into each of the 6 microfuge tubes. 17. Add 20 µl of the phage suspension to the first, mix and transfer 20 µl to the next tube. 18. Continue this operation until the last tube. Remember to change tips at every dilution! 19. Save the remaining lysate at 4°C. 20. Titer the phage to determine the number of phage particles in your lysate:
To do this: 1. Place 100 µl of fresh culture of E. coli KL227 in 3 x 15 ml tubes. Add 10 µl CaCl2 (from a100mM stock
solution, before adding 100 µl of your phage). 2. Add 100 µl of P1Cm c1-100 dilutions 10-4, 10-5, 10-6 to individual tubes. 3. Incubate the tubes at 30°C for 10 minutes for adsorption of the phage. 4. Label 3 R-Base agar plates. 5. Only when you’re ready. Add 4ml of melted R-top agar to the tubes with bacteria – phage dilution tubes. 6. Mix the tubes by rolling them between your palms. 7. Pour the melted agar onto the plates. Act fast before the agar has a chance to solidify! 8. When the agar has solidified, invert the plates as usual and incubate at 40°C. Prepare your rif (rpoB) mutant for transduction: Inoculate 2ml LB rif with rifR (rpoB) mutant and incubate at 37°C with shaking overnight.
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Experiment II: Day 6 Worksheet. To be completed during the lab period. 1. Why did we grow the lysogen and lyse the cells last lab? (What were we trying to obtain?) 2. Why do we add chloroform to the lysate? 4. Why are we adding E. coli KL227 to the lysate tubes for titration of the phage? 5. Why are we plating three dilutions of phage for titration? 6. Why do we need to calculate the titer?
Name:
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Experiment II Day 7 (10/4): Transduction 1. Count the plaques on your titration plates.
Note: P1 plaques are very small and not easy to distinguish; use magnifiers if necessary. 2. Calculate the titer of the phage using the dilution you trust most. 3. Put 200 µl of the rifR (rpoB) mutant of E. coli HR171 his- argH- trp- into 2 microfuge tubes. 4. Add 108 PFU to one tube. 5. Add 100 mM CaCl2 to a final concentration (f.c.) of 5 mM Note: You should use the C1V1 = C2V2 equation to determine the volume of calcium chloride to add. You
will need to add these volumes based upon the unique volume of phage you already added. The tubes will have a different V2; right?!
6. Incubate tubes at 30 oC for 10 minutes.
7. Prepare two plates: 8. Spread 100 µl each trp, his, met, on both minimal media plates. 9. Add sodium citrate to tubes a f.c. of 10mM.
Note: You should be able to calculate this now. Be sure you check C1. 10. Centrifuge tubes for 20 seconds. 11. Decant the supernatant. 12. Resuspend in remaining supernatant. 13. Spread the whole volume on plates. 14. Incubate the plates at 30°C.
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Experiment II: Day 7 Worksheet. To be completed during the lab period. 1. The tube without phage is a control for what? 2. Why do we not use arginine on the plates? 3. Next lab we’ll patch the transductants on minimal media with different additions. Which plate is the permissive plate? 4. Given that sequencing of the E. coli genome is cheap and easy, what is the value in learning of these gene transfer techniques? Or, aside from mapping, what are other uses of conjugation and transduction?
Name:
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Experiment II Transduction: Day 8 (10/6) Analysis of the Transductants 1. Using a toothpick patch the transductants as follows: 2. Incubate the plates at 30°C.
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Experiment II Transduction: Day 9 (10/11) Analysis of the Transductants
1. Record and analyze your transduction data as well as the pooled class data. 2. Perform the 3-point cross analysis. Note: KL 227 (donor) = argH+ metA- rifS rifR mutant of HR171 (recipient) = argH- metA+ rifR
Recombinant Phenotype Number of Recombinants
arg+ met+ rifR
arg+ met+ rifS
arg+ met- rifR
arg+ met- rifS
Draw the recombination crossovers to help you determine the order of arg, met and rif genes on the chromosome.
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Questions to answer in your Report on Experiment II: Mutations in Bacteria
1. You plated 1ml of an overnight culture (O/N) of HR171 on your LB rifampin plate. A typical O/N contains about 5 x 109 bacteria/ml. After incubation how many colonies did you observe?
2. What was the apparent frequency of rifampin resistant mutants in the sample you plated? 3. Would you expect the fraction of bacteria that are spontaneous rifampin resistant mutants to be higher
or lower in a culture you’ve propagated many times or one you had just started from a single colony of sensitive bacteria? Why?
4. What would you expect to be more frequent in a culture you have propagated, Met- mutants or rifampin resistant mutants? Why? Note: The culture was grown in media with methionine and without rifampin.
Extra credit: 5. Rifampin inhibits the β subunit of the E. coli RNA polymerase. A typical rifR mutant contains a mutant
gene that produces a β subunit that no longer binds rifampin. Propose one other genetic alteration by which an E. coli might become resistant to the drug rifampin (without mating with other bacteria) and the molecular basis for this other resistant phenotype. What is your evidence this other type doesn’t exist?
Conjugation
6. In your Hfr cross, why didn't the KL16 strain grow on the selective plates? 7. In your cross, were there more His+ recombinants than Trp+ or Arg+ recombinants? If so, why? 8. Would you expect more of the Arg+ recombinants to be rifampin sensitive than either the His+ or Trp+
recombinants? If so, why? 9. Were any of the His+ recombinants also Trp+? Were any Arg+? Were any rifampin sensitive? Plot
your data to see if it agrees with the expected results for where the rifampin resistance mutation lies. Transduction
10. You transduced your rifR HR171 with P1 grown on KL227 metA [Met-]. You selected Arg+ transductants. For the class as a whole, what percentage of Arg+ transductants were Met-, what percent were rifS? Can the rif and arg markers be co-transduced with the met marker? If so what is the order of these three genes from the combined data? Is this consistent with which recombinant type is the rarest from the three factor cross? How can you tell?
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Experiment III: Transposon Mutagenesis and Gene Mapping
Transposable elements (also known as transposons) are small DNA fragments that can move from one site in DNA to another. Transposons can insert into new locations throughout the DNA of the bacterial cell in which they are present. Some transposable elements can integrate almost anywhere in the genome including into the middle of any pre-existing gene. When this happens the transposon almost always inactivates the gene thereby creating a mutant. Of course, if the gene is an essential one (and since genes are haploid in bacterial cells) this will kill the bacterium, and we will never be able to isolate this particular mutant. Often, however, the integration will be in a non-essential gene whose mutant phenotype can be detected.
Transposon-generated mutations are especially useful because the transposon then marks the mutant gene both genetically and physically. It marks the mutant genetically if the transposon contains a detectable gene. The transposon-generated mutant gene can then be followed by mapping the location of that gene. The transposon marks the gene physically because the DNA of the transposon then exists as a several thousand base pair insertion into the DNA of the mutated gene. The size and the nucleotide sequence of the transposon DNA are already known for many transposons. A partial sequence of the gene in which the transposon has inserted can, therefore, be determined by sequencing from a primer complementary to a sequence near the end of the transposon.
To isolate bacteria in which a transposon has inserted into the chromosome the transposon is introduced into the cell on a DNA that cannot replicate in that cell (a “suicide vector”). We shall use as a suicide vector, a plasmid (pSAM_DKm) that contains the origin of replication of an E. coli plasmid R6K, but not its replication protein called π (pi), encoded by the pir gene. This plasmid can replicate only in bacterial cells that were engineered to carry the pir gene. If pSAM_DKm plasmid is introduced into a cell that does not carry the pir gene the only way the cell can become resistant to kanamicin (carried by the transposon) is if the transposon is transferred into another self-replicated DNA molecule in the cell (another replicon), either the chromosome or a plasmid.
In this experiment, you will insert the Mariner-Km transposon into the chromosome of the P. putida strain 2440 and screen for mutations that will inactivate the ability of this strain to use levulinic acid as the carbon and energy source. To date these genes are unknown. Indications have been obtained pointing to the existence of several such genes. The discovery of all such genes will help to understand how P. putida manages to use this unusual carbon source in contrast to many other bacteria.
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Experiment III: Day 1 (10/13) Purification of Plasmid Containing the HiMar-Km Transposon A culture of the strain CB2870 carrying the plasmid pSAM_DKm which contains the HiMar-Km transposon will be ready for you. Use it to purify the plasmid by the following method: Qiagen DNA Prep Protocols for 4.5 ml Cultures
1. Overnight cultures of 5ml in LB medium + Km + Amp + DAP (0.3mM). 2. Centrifuge three 1.5 ml aliquots in 2ml microfuge tubes for 30s -1 min at maximum speed. 3. Discard the supernatant carefully (keep the pellets) and resuspend each pellet in 0.5 ml STE buffer. 4. Combine the suspensions in one microfuge tube 5. Centrifuge as in step 2 and carefully discard the supernatant (make sure that pellet does not come loose) 6. Suspend pellet in 250µL Buffer P1 (kept cold). 7. Add 250µL Buffer P2 (Lysis Buffer). Mix by inversion 10 times. Do not vortex!
Do not exceed 3 min of the lysis. 8. Add 350µL Buffer N3. Mix well by inversion as above. Do not vortex. 9. Incubate on ice 5 min.
10. Centrifuge at maximum speed for 10 min. Team up for the use of the centrifuges. Supernatant should be clear.
11. Transfer the supernatant onto the Spin Column. 12. Centrifuge at 8,000 x g for 1 minute. 13. Discard the flow through. 14. Wash column with 100µL Buffer PB, centrifuge as in step 12. Discard flow through. 15. Wash column with 750µL Buffer PE, centrifuge as in step 12. Discard flow through. 16. Centrifuge the empty column for an additional 1 min at 8,000 x g. 17. Transfer column to a clean 1.5ml microfuge tube. 18. Add 50µL buffer EB to the center of the column. 19. Wait 5 min. 20. Centrifuge as in 16. 21. Save the flow through and store at 4°C.
This is your purified plasmid DNA to be used for transposon introduction into the P. putida strain.
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Experiment III: Day 1 Worksheet Name: To be completed during the lab period.
1. What happens upon addition of the Lysis Buffer (P2) to bacterial cells?
2. Why is it so important to NOT vortex at various steps of plasmid DNA purification?
3. Why should we not exceed 3 minutes for the lysis?
4. What happens upon addition of the Neutralization Buffer (N3) to the lysate?
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Experiment III: Day 2 (10/18): Concentration of DNA by Ethanol Precipitation
1. Add 0.1 volume of 4M sodium acetate solution to your DNA solution (for volume, see day 1, step 18). What is a ‘volume’?
2. If available, add 1µl of “*Pellet Paint”. *Note: This may already be added to the ethanol for you for you. If this is the case vortex the ethanol solution before using in point 3. since the Pellet Paint would have precipitated.
3. Add 2 volumes of absolute ethanol. Mix well. 4. Mark the side of the tube where you expect the precipitate. 5. Centrifuge at max speed for 10min. 6. Decant the ethanol. 7. Wash the precipitate with 400µl of 70% ethanol. 8. Centrifuge as before. 9. Decant the ethanol.
10. Leave the tube opened to dry the ethanol for at least 20 min. Note: If the SpeedVac air dryer is available, you may use it to decrease your wait.
11. Dissolve the precipitate in 15µl of dH2O. 12. Store at 4°C.
This is your DNA of the pSAM_DKm plasmid – the donor of the HiMar-Km transposon.
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Experiment III: Day 2 Worksheet. To be completed during the lab period. 1. Why are we adding sodium acetate before ethanol precipitation of DNA? 2. Describe what ethanol does to DNA. 3. Why must we be careful to allow all the ethanol to evaporate before dissolving the DNA precipitate? 4. Why must we be sure to dissolve our precipitated product in only water?
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Experiment III: Day 3 (10/20): Electroporation of Pseudomonas putida (strain 2440).
Note: Each student will prepare two tubes that are combined in step 4. One student per bench will prepare a second set of two tubes to serve as a control. Do not add DNA to the control tube. Protecting sterility in the following procedure is of utmost importance. 1. Place 1.5ml of the overnight culture of P. putida strain KT2440 into each of two 2ml microfuge tubes. 2. Centrifuge 1 min at 7,000 x g. 3. Wash twice with 300µl 300 mM sucrose each.
Note: Make sure that the pellet is completely resuspended each time. 4. Suspend each portion in 80µl 300mM sucrose and combine. 5. Add 100µL culture to your 15µl of dissolved pSAM_DKm plasmid DNA (directly into cuvett) 6. One person per bench uses, in addition, 100µl of the electro-competent culture without DNA. This is
your control. 7. Transfer to electroporation cuvette. 8. Electroporate each at 2.5kV, 25µF 200Ω in 2mm gap cuvette. Record reading (≈ 4.7 - 4.8 ms). 9. Add 1ml LB to cuvette.
10. Pour contents into a 2 ml microfuge tube. 11. Shake for 1.5hrs at 32°C. 12. Plate 0.1ml of each onto a M9 + glucose + Km(100µg/ml) plates. 13. Save your culture for the next class. It can be concentrated and replated if there are too few colonies on
the first plate. If this is the case centrifuge the suspension as in 2. 14. Decant supernatant. 15. Resuspend the pellet in the medium remaining in the tube. 16. Plate entire volume onto another M9 + glucose + Km plate. 17. The persons preparing control samples plate the concentrated control onto one M9 + glucose + Km plate 18. Incubate the plates at 32° C for 2 days.
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Experiment III: Day 3 Worksheet Name:
To be completed during the lab period.
1. Why are we incubating the culture after the electroporation in a medium that does not contain antibiotic?
2. What is going to happen in the cell after the electroporation?
3. What is the control ‘controlling’ for?
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Experiment III: Day 4 (10/25) Screening for Mutants
1. Examine the results of your electroporation plates. Count the colonies. 2. Mark plates containing minimal agar + levulinic acid and minimal agar + glucose at the top. 3. Use the papers with the grid provided to you. 4. Using a toothpick or a wire, patch colonies onto plates as follows. Be careful to preserve sterility. 5. Repeat such that you screen at 100 - 200 colonies. In this type of procedure the more is the better! 6. Incubate the plates at 30°C for 2 days. Experiment III: Day 5 (10/27): Purification of Mutants 1. Identify colonies that grow on glucose, but not on levulinic acid plates. These are, potentially, your
mutants. 2. Divide an M9-glucose and an M9 levulinik acid plate into 4 quadrants each. 3. Streak one of your mutants per quadrant. Share plates if necessary.
Note: When you streak bacteria you should always streak for single colony isolation. 4. Use one plate for 4 different colonies. If you don’t have a mutant of your own find someone that has two
mutants and use one of those. Alternatively, use one of your colleagues’ mutants, but keep track on which one it is.
5. Number the colonies with consecutive numbers throughout the entire class so we can identify them after sequencing. Make note of your mutant number for your report.
6. This assay verifies that you have indeed a mutant unable to grow on levulinic acid. 7. Incubate plates at 32°C. Experiment III: Day 6 (11/1): Culturing of the Transposon Mutants
1. Identify a well-isolate colony of the mutant unable to grow on levulinic acid. 2. Inoculate 4ml LB. 3. Incubate at 32°C overnight.
Note: it is CRITICAL that you use an isolated colony and that you record the mutant number(s).
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Experiment III: Day 7 (11/3): Isolation of chromosomal DNA (Promega Kit)
1. Centrifuge 1 ml of overnight culture at 13,000–16,000 × g, for 1 min (use 1.5ml microfuge tube). Discard the supernatant (repeat if small cell pellet).
2. Add 600µl Nuclei Lysis Solution. Pipet gently to mix. 3. Incubate for 5 minutes at 70°C, then cool to room temperature. 4. Add 3µl of RNase Solution. Mix, incubate at 37°C for 45 minutes, then cool to room temperature.
Protein Precipitation 5. Add 200µl of Protein Precipitation Solution. Vortex. 6. Incubate on ice for 5 minutes. 7. Centrifuge at 13,000–16,000 × g for 3 minutes.
DNA Precipitation and Rehydration 8. Transfer the supernatant to a clean tube containing 600µl of room temperature isopropanol. Vortex. 9. Centrifuge as in step 7 (see above). Discard supernatant. Mark the side where you expect the
precipitate. 10. Add 600 µl 70% ethanol. Mix. 11. Centrifuge as above. Discard supernatant. Aspirate the rest of the supernatant or blot it on absorbing
paper until all ethanol is removed. 12. Air dry the precipitate for 15 min. 13. Rehydrate the DNA pellet in 100µl of Rehydration Solution for 1 hour at 65°C or overnight at 4°C. Experiment III: Day 8 (11/8) First round of PCR for lev mutants Mix for the control DNA:
Nr DNA µl Random Primers (20pmol/µl) µl Polymerase mix µl
1 2 3 1a 1 KT2440 1 - 1 - - 23 2 KT2440 1 - - 1 - 23 3 KT2440 1 1 23
The DNA of KT2440 is your control that does not contain the transposon. This mix is prepared one per bench. Mix for the mutant DNA:
Nr DNA µl Random Primers (20pmol/µl) µl Polymerase mix µl
1 2 3 1a 4 lev # 1 1 - 1 - - 23 5 lev #1 1 - - 1 - 23 6 lev #1 1 - - - 1 23
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Polymerase mix Round 1 for 10 reactions. To be recalculated according to the number of mutants to be tested. This mixture will be prepared by the instructors and added to the wells.
µl Final conc
10x Dream Taq Buffer 25.0 1 dNTP 10 mM 5.0 0.2 mM Round1-pSAM_DYH (20pmol/ul) 10.0 0.8 pm/µl Fermentas Dream Taq Polymerase (5u/µl) 3.0 1.7 u H2O 187.0
Thermocycler program 12: Step 1 94oC 2 min Step 2 94oC 30 sec Step 3 50 oC 40 sec *reduce temp by 1 oC for each subsequent round Step 4 72 oC 3 min Step 5 Go to Step 2: 10X (this is the touchdown phase) Step 6 94 oC 30 sec Step 7 62 oC 40 sec Step 8 72 oC 3 min Step 9 Go to Step 6: 25X (this is the amplification phase) Step 10 4 oC Hold The PCR reactions will be run in a 96 well PCR plate. It is ESSENTIAL that you make note on the coordinates where you have placed your mutant DNA.
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Primer Sequences
Round 1-RndomPA-1 5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNCGATG-3' PA genomic random primer for first round PCR
Round 1-RndomPA-2 5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNGGTAG-3' PA genomic random primer for first round PCR
Round 1-RndomPA-3 5'-GGCCACGCGTCGACTAGTACNNNNNNNSNSSCGAAG-3' PA genomic random primer for first round PCR
Round1-pSAM_DYH 5'-TCGAGGGCGCGCCAAGCAG-3'
Transposon primer for first round PCR for semi-random PCR; specific to the end of
pSAM transposons
Round2-PA 5'-GGCCACGCGTCGACTAGTAC-3' The second round PCR Primer that is anchored to the Round
1-RandomPA primers
Round2-pSAM_DYH 5'-CGCGCCAAGCAGAAGACGGCATACG-3'
Transposon primer for the second round PCR for semi- random PCR; specific to the end of pSAM transposons
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Experiment III: Day 9 (11/10) Round 2 PCR for the mutants
Polymerase mix Round 2 for 10 reactions. To be recalculated according to the number of mutants to be tested. This mixture will be prepared by the instructors and added to the wells. µl Final Conc. 10x Dream Taq Buffer 50.0 1 dNTP 10 mM 10.0 0.2 mM Round2 SAM DYH (20 pmols/µl) 14.0 0.56 pmol/µl Round2 PA (20 pmols/µl) 14.0 0.56 pmol/µl Fermentas Dream Taq Polymerase 6.0 1.7 u/tube H2O 366.0 46.0 µl per tube + 4 µl of the Round 2 PCR mix Experiment III: Day 10 (11/15) Separation of DNA fragments by electrophoresis
1. Mix 5 µl of your amplification mix, and place in the slots of a 1% agarose gel. Instructors will apply molecular size standards on each of the first slot of the gel.
2. Run electrophoresis for 1 hour, at 100 volts. 3. Photograph the gel in UV light. Instructors will demonstrate the use of the camera. 4. Identify the bands strong enough to be purified and sent for sequence determination.
Experiment III: Day 11 (11/17) Preparative Separation of DNA fragments by electrophoresis
1. Add 8 µl of a “Stop Buffer” and place the mixture into two slots of a 1% agarose gel approximately half into each slot. Try to use all the mixture you have.
2. Run electrophoresis for 1 hour. 3. Cut out the strongest band under UV light (Protective goggles!) 4. Place into a preweighed 2 ml microfuge tube 5. Determine the weight of the gel slice.
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Experiment III: Day 12 (11/22) Extraction of DNA from the gel
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl).
For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved).Vortex the tube every 2–3
min to help dissolve gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to
Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M
sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30 sec at approximately
8,000 x g. Discard flow-through and place the QIAquick column back into the same tube. For sample
volumes of >800 µl, load and spin again.
8. Add 0.5 ml Buffer QG to the QIAquick column and centrifuge as before. Discard flow-through and
place the QIAquick column back into the same tube.
9. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge s before. Discard flow-through and
place the QIAquick column back into the same tube.
Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing blunt-ended
ligation), MUST let the column stand 2–5 min after addition of Buffer PE.
10. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g
(13,000 rpm) to remove residual wash buffer.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 30 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick membrane,
wait for 5 min and centrifuge the column for 1 min as in 10.
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Experiment III: Day 13 (11/29): Concentration of DNA by Ethanol Precipitation
This is performed as described in the Experiment III: Day 2 Dissolve the final DNA precipitat in 12 µl of water. Check Nanodrop. If your DNA conc. > 5 ng/µl, then dilute it to ≈ 2 ng/µl (most likely no one should have to dilute). To prepare the DNA for sequencing add 10 µl of DNA and 1 µl of the primer Round2 SAM DYH or primer Round2 PA. Send the plate for sequence determination. Record plate coordinates!
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Questions to answer in your final report on Experiment III
1. How was the transposon, SAM_DKm-Km, introduced into the Pseudomonas cells?
2. How was it ascertained that the colonies you obtained after electroporation contain transposons inserted into the chromosome of the cells?
3. Which types of mutants did you look for? How were they found?
4. How were the genes, affected by transposon insertions, identified?
5. You were told that a specific primer was going to be added to your DNA sample before sequencing, what do you suppose is the target of primer? How will this help us determine which gene or genes are responsible P. putida's ability to use levulinic acid as a carbon and energy source?
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6. Your lab partner (Aaron) wasn't paying attention when the instructors were going over how to determine
if you had enough DNA in your sample for sequencing! Explain to Aaron how he can calculate the total amount of DNA in his sample if the Nano-vue spectrophotometer said his concentration was 81.5 ng/µL, (he forgot his calculator too, so be a good lab mate and do the calculations for him) he now has a total volume of 28 µL. If Aaron has enough DNA in his sample, figure out the volume he would need to concentrate his sample to get a concentration of 200 ng/µL.
7. Extra credit: explain why the defect in the gene you worked with resulted in this particular phenotype.
