Chp3 PGLOMutagenesis

pGLO Mutagenesis Session 2Content objectives:

To understand how mutations can alter gene expression. To understand the principles of bacterial isolation. To understand how restriction enzymes function.

Scientific method objectives: To perform data analysis. To use computer simulation to design an experiment.

Laboratory skill objectives: To demonstrate skill in aseptic technique and bacterial isolation.

Preparation and AssignmentsBefore lab:NOTE: For each laboratory session you will be expected to have read and understood the material for the day’s lab session and have taken notes on any procedural videos before arriving in class.

Read this lab manual chapter Complete the pGLO: Session 2 Pre-lab assignment, which is due at the

start of class

During class: Review the pGLO: Session 2 Pre-lab assignment Answer verbal questions posed by your Laboratory Instructor Design your restriction digestion experiments using computer simulation. Start your pGLO: Session 2 Post-lab assignment.

After class: Complete your pGLO: Session 2 Post-lab assignment- This assignment is

due at the start of pGLO: Session 3.

Use of any section of this Lab Manual without the written consent of the Department of Biology, University of Pennsylvania is strictly prohibited.

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IntroductionIsolating Mutants

In this lab, your goal is to isolate E.coli cells expressing the G- mutant phenotype, i.e. those cells that do not glow in the presence of arabinose. In the last session, you transformed competent E.coli cells with wild-type (Wt) or mutant (M) pGLO plasmids and plated the transformed cells on selective media. In this session you will look at these plates to find the G- mutant phenotype. It is necessary that you isolate G- mutants alone and not E.coli strains that may carry more than one mutation in the pGLO plasmid (especially double mutants that could also have an insertion in the ampicillin resistance gene).

Patching

There are many ways of isolating mutants. Patching is the technique of using a sterile toothpick to lift a whole bacterial colony off an agar plate and transferring it to the surface of a fresh agar plate. Patching can do 2 things for you: verify the phenotype of mutants and give you enough DNA to work with for subsequent analysis. In this session, you will use patching to 1) verify the phenotype of your G- mutants and 2) produce enough cells so that you have sufficient copies of your mutated pGLO plasmid DNA for restriction digestion and sequencing analysis. In the next parts of the experiment, you will use restriction digestion and sequencing analysis to determine where exactly the EZ-Tn5 transposon has inserted into the pGLO plasmid.

Restriction Digestion

Remember that E.coli cells with the G- mutant phenotype contain a pGLO plasmid with a transposon insertion in either the araC or GFP genes. You would like to know which of these two genes has been mutated but you cannot tell that by examining the phenotype alone. One way to determine where the insertion has occurred is to first isolate the mutated pGLO plasmid DNA from the E.coli cells you will patch today (you will do this next week in pGLO: Session 3) and then use restriction digestion analysis to determine whether the araC gene or GFP gene contains the EZ-Tn5 transposon insertion (you will do this in pGLO: Session 4). It is important that you design an appropriate restriction digestion experiment so that you will be able to determine which of the two genes, araC or GFP, has been mutated. In today’s lab session you will perform a simulated digestion of the wild type pGLO plasmid using computer software to determine which restriction enzymes you should use to digest your mutant pGLO plasmid DNA so that you separate the araC gene from the GFP gene.

Restriction enzymes (REs), produced by microorganisms, are enzymes that recognize a short specific base sequence in a DNA molecule and cleave the DNA at that point. Microorganisms that produce REs can immediately digest a foreign piece of DNA once it enters the cell if the DNA carries the base sequence specific for those REs. This is a major defense mechanism that many bacteria use for the destruction of invading viral DNA.


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Because this foreign DNA is “restricted from action in these bacteria” (i.e. it can no longer function within the host cell because it has been cut) the term "restriction enzyme" or "restriction endonuclease" is used to describe this class of enzymes.

You may ask yourself why the REs produced by a microorganism do not digest its own DNA? The reason is that REs recognize only specific short sequences of bases in the DNA molecule and either the host DNA does not contain the these sequences or if such sequences exist, they are modified in a way that the REs cannot recognize them. The most common type of modification is the addition of a methyl group to one or more of the bases of the recognition sequence. Methylation does not alter the coding capacity of the DNA.

The discovery of REs and the mode of their action paved the way for molecular cloning and genetic engineering. In 1970, Hamilton Smith was the first to isolate an enzyme called HindII from Haemophilus influenza, which could cut the DNA of phage T7 (a virus) into 40 specific fragments. When the ends of the DNA fragments from the digested phage T7 DNA were sequenced, it was found that all fragments had either a "C-A-Pu" or a "Py-T-G" (Pu stands for purine and Py for pyrimidine). Smith concluded that HindII acted at a very specific point on the DNA molecule as is shown below:

◊ 5' •••-G-T-Py-Pu-A-C-••• 3' | | | | | | 3' •••-C-A-Pu-Py-T-G-••• 5' ◊

In the above diagram, shows the site of action of the enzyme. Interestingly these ◊sequences also exist in the host Haemophilus influenzae from which the HindII was isolated; however, the adenines are methylated and as such HindII is not able to recognize the sequence in the host.

Another example of a common RE is EcoRI, which as the name indicates, is produced by E. coli. The gene for this RE is on an R plasmid (a plasmid conferring resistance to antibiotics). Its cleaving action is as follows:

◊ 5' •••-G-A-A-T-T-C-••• 3'

| | | | | | 3' •••-C-T-T-A-A-G-••• 5' ◊

This sequence exists in the virus SV40 and also in the E. coli itself, but again in E. coli one of the adenine bases on each strand is methylated.

It is apparent from these two examples that no matter which strand is cut with the RE, the sequence of the bases at the ends are identical if you turn one fragment 180° over the other. (This is due to the palindromic--twofold symmetrical--nature of the sequence of bases on which REs are active.) Note that in HindII the point of action of the RE ( ) on the ◊


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two strands coincides while for EcoRI, the points are 4 bases apart. Since the two strands of the DNA stay together, HindII produces clean cuts called "blunt ends" while EcoRI produces staggered cuts or 5'-overhanging ends called "sticky ends". In both of the examples above, the recognition sequences are 6 bases long; however, many REs can recognize specific sequences of from 4 to over 20 bases. On the average, REs that act on fewer bases cut a DNA molecule more times than those that act on longer base sequences. Finally, REs are commonly named using 3-4 letters and a Roman numeral. The letters designate the microorganism from which the enzyme was isolated and the Roman letter indicates different enzymes from the same source.


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pGLO Mutagenesis: Session 2 ProcedureOverview

In this lab session, students will examine the results from the experiment they conducted in pGLO: Session 1. This data will be recorded in their lab notebooks and also used to answer the questions in the pGLO: Session 2 post-lab assignment. Each student pair will then patch G- mutant colonies onto selective media. Once patching is complete, students will work in pairs to design a restriction digestion experiment using computer simulation software. Students will submit this information to their Lab Instructor or TA before leaving lab so that we may order all necessary REs the students would like to use in pGLO: Session 4.

Materials needed

Sterile blunt end toothpicks L+Kan+Ara+Amp media plates (1 plate per pair of students) UV lights Protective eyeglasses Sharpie pens in different colors for counting colonies Laptops NEB Catalog RE Inventory List

Procedural Notes

Data Analysis of pGLO: Session 1 Experiment

1. Examine the plates that you made in pGLO: Session 1. Document the results from these plates, including counts of glowing and non-glowing colonies, in your lab notebook and in the pGLO: Session 2 post-lab assignment. Discuss whether or not these results make sense. Make sure to share your data with the other groups at your lab table.

When counting glowing colonies, please follow theses directions: -Put on protective eye goggles and turn on "long wave" UV lamp. -This procedure works best if you have a dark background behind the plates so turn off the lights in the classroom and/or move to a dark place in the room. -Hold the lamp over the inverted plates and move it to make sure you cover all parts of the plate. -Do not forget to turn off the lamp after you are finished.


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2. Once all data has been collected, discard the wild-type pGLO plates ONLY in the biohazard bins located at the front of the room.

3. Keep the mutant pGLO plates. These are the plates you will use for patching.

Isolation of G- Mutants via Patching

1. Obtain a new L+Amp+Kan+Ara plate and label it (on the bottom near the edge) with all necessary identifying information (Section #, group name, media, pGLO 2).

2. Wearing goggles and using the UV lamp, circle the G- mutant colonies with a fine-point marking pen on the mutant pGLO plates (be sure you DO NOT use the positive and negative control plates from the mutant pGLO screen for this activity). These will be the colonies you will patch in today’s lab.

3. Each pair of students will need a minimum of 2 unique G- colonies for the next session, however it is strongly encouraged that you patch as many G- colonies as possible from the experimental plates (up to 20 mutants/pair). If you have very few G- colonies, you may borrow some from other groups that have them in abundance.

4. Watch the demo by your Lab Instructor or TA on how to patch G- colonies onto your new L+Amp+Kan+Ara plate.

-To make patching easier, ask your Lab Instructor or TA for a grid that you can place under the new L+Amp+Kan+Ara plate.

-Be sure to use a fresh toothpick for each G- colony and discard the used toothpicks in the waste beaker on your lab bench.

5. Invert the newly patched plate and incubate it at 37°C for 40-48 hours. Your Lab Instructor or TA will store them for you in the lab refrigerator until the next session.

6. Record the procedure you used for isolating G- mutants via patching in your lab notebook.

Restriction Digestion Simulation

1. Working in pairs, open your browser and go to the NCBI site at http://www.ncbi.nlm.nih.gov . In the search box, change the drop down menu from "All Databases" to "Nucleotide" and in the lower box, type the formal name of the pGLO plasmid, "pBAD-GFPuv", and then click on "Search".


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http://www.ncbi.nlm.nih.gov/

2. A new page will appear that contains information about the pGLO sequence. Look through the data carefully and note the size of the plasmid, the genes present, the location of each gene on the plasmid and the sequence of bases.

3. Write down the GenBank number for the “pBAD-GFPuv” entry here:

a. GenBank number: _____________________________

4. We will be purchasing your restriction enzymes from the company New England Biolabs. This company provides an online tool to run simulated restriction digestions. Open this website at http://tools.neb.com/NEBcutter2/.

5. Enter the GenBank number for pBAD-GFPuv plasmid in the appropriate box.

6. Towards the bottom left of the gray box select: “This sequence is CIRCULAR” The select “Submit” on the right hand side of the gray box.

7. A map of the wild-type pGLO plasmid with several (but not all) restriction enzyme cut sites will appear once the simulated digest is complete. Fill in the following information as it pertains to this map:

a. What gene is represented at position “a”? ________________

b. What gene is represented at position “b”? ________________

c. What gene is represented at position “c”? ________________

8. You want to design a restriction digest experiment that will separate the araC gene from the GFP gene. Looking at the map of pGLO and the list of REs available for you to use in your experiment (ask for this list from your Lab Instructor or TA), select 2-3 enzymes that might be good for you to use in your experiment. Note this map will show you several, but not all of the restriction enzymes that cut the pGLO plasmid. To see a full list of enzymes that cut the pGLO plasmid select “Custom Digest” at the bottom left-hand part of the page.

9. Once you have selected the restriction enzymes you want to use in your experiment you can now perform a custom digest of the pGLO plasmid using only these enzymes following the instructions below.

10. First, select the statement towards the top of the new page that says “Enzymes with compatible buffers.” Check to see if the enzymes you would like to use are grouped under a single buffer type (either the CutSmart Buffer, NEB Buffer 1.1, NEB Buffer 2.1, or NEB Buffer 3.1). If any of these enzymes are not found in the same buffer system as the other enzymes you


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http://tools.neb.com/NEBcutter2/

have selected, you will need to select a different set of enzymes as you can only add one buffer solution to your restriction digestion reaction.

11. Once you have selected enzymes that are grouped under a single buffer type, you will need to check if each enzyme has 100% activity in that buffer system. If any of the enzymes do not have 100% activity you will need to select a different set of enzymes as it is critical that the all the enzymes in the digestion reaction work with 100% activity in the same buffer system.

12. Once you have selected 2-3 enzymes with a 100% activity in a single buffer system, click on the boxes next to the enzyme names and select the green “Digest” button at the bottom of the page.

13. Examine this new pGLO map that shows only your selected restriction enzymes’ cut sites and confirm that the enzymes you have selected would separate the araC gene from the GFP gene.

14. Towards the bottom left-hand side of the page that has your new pGLO map you will find the selection “View gel.” Select this button.

15. This new page will show you the # of DNA fragments that would be produced by this digest (upper right-hand side) and their lengths. Note the # of fragments that would be produced and their lengths. Based on this information answer the following questions: 1) Is there more than 1 fragment for any given length? and 2) do any of the fragments differ in length by less than 200bp? If the answer is yes to either question you will need to redesign your experiment. Discuss why you would need to redesign in these cases with your Lab Instructor or TA.

16. Finally, you need to confirm that the enzymes you have selected will not cut the EZ-Tn5 transposon DNA that has been inserted into either of these genes. To do this first go to http://www.epibio.com/tech-support/dna-sequences

17. Select “EZ-Tn5™ <Kan-2> [1221bp]” link. When the sequence appears on the next page, select “Text File” at the top of the page.

18. When the text file of the sequence appears on the next screen, copy the EZ-Tn5 sequence.

19. In a new tab, open NEB Cutter at http://tools.neb.com/NEBcutter2/ and paste the EZ-Tn5 sequence into the white box. Select “This sequence is LINEAR” and and then select “Submit.”

20. This new map will show several (but not all) restriction enzyme cut sites in the EZ-Tn5 DNA sequence. To see a full list of enzymes that cut EZ-Tn5 select “Custom Digest” at the bottom left-hand part of the page.


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http://tools.neb.com/NEBcutter2/

http://www.epibio.com/tech-support/dna-sequences

21. Scroll through the list of enzymes. If any of the enzymes you would like to use in your experiment appear in this list (and thus cut the EZ-Tn5), you will need to redesign your restriction digestion.

22. Now that you have selected 2-3 enzymes that work with 100% activity in the same buffer system, produce DNA fragments of unique lengths that are at least 200bp different in size, and do not cut the EZ-Tn5 DNA, you should plan how you will actually carry out the experiment in the lab. Using the NEB Catalog, look up the information below for each enzyme that you are proposing to use in your experiment. Based on these answers, you may wish to redesign your restriction digestion experiment to make it logistically feasible for pGLO: Session 4.

a. What is/are the incubation temperate(s) and lengths of incubation for your enzymes?

b. Do any of the enzymes require heat deactivation? If so, what is/are the incubation temperate(s) and lengths of incubation for deactivation of your enzymes?

c. Do any of your enzymes require the addition of BSA? Which ones?d. Draw a timeline for your experiment that includes the order in which

enzymes should be added to the reaction (and if they require the addition of BSA), the temperatures & lengths of incubation, and the temperatue and lengths of incubation for deactivation.

e. How long will your experiment take to complete? (If the answer is more than 2 hours, you will need to redesign your experiment)

23. Once you feel you have successfully designed your restriction digestion experiment, answer the questions in the pGLO: Session 2 post-lab assignment. Make sure to share this information with your Lab Instructor or TA before you leave the lab today. We will order the restriction enzymes you are requesting and have them available for your use in pGLO: Session 4.

Please note: Failure to share your restriction digestion experiment with your Lab Instructor or TA before leaving today may result in our inability to acquire the enzymes you need in time for pGLO: Session 4. This situation will have a negative impact on your grade.


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Name: ____Sebastian de Armas___ BIOL123-Fall 2014 Section # __105_

pGLO: Session 2 Pre-lab Assignment Due at the start of pGLO: Session 2

1. (1.5pts) One of the main objectives in pGLO: Session 2 is to isolate G- mutants from the plates you made in pGLO Mutagenesis: Session 1. How do you propose to do this in today’s lab? We are looking for plates where the cells were resistant to both kanamycin and ampicillin, and although being in the presence of arabinose did not glow because of the transposon inserting itself into the GFP DNA sequence

2. (0.5pts) What safety precautions do you need to take in today’s lab when working with the UV lamps?Do not keep your body in the way of the UV rays for too much, wear gloves and covering to protect from radiation. We will also need protective eye goggles.

3. (1pt) What are restriction enzymes?Restriction enzymes are enzymes that are able to locate and cut away different (specific) pieces of DNA away from a DNA strand through splicing. They are enzymes that recognize a short specific base sequence in a DNA molecule and cleave the DNA at that point

4. (1pt) How do restriction enzymes aid in microbial survival?

Microorganisms that produce restriction enzymes can digest foreign pieces of DNA once it enters the cell if the DNA contains the specific sequencing that the restriction enzyme is looking for This is a huge defense mechanism that many bacteria use for the destruction of invading viral DNA (it essentially creates a specific immune system).

5. (1pt) List two mechanisms for how microbes protect themselves against the activity of their own restriction enzymes?

Restriction enzymes only recognize short sequences in the DNA molecule, and the case is usually that the host organism does not contain those specific base pairings in its own genome or if they exist the organisms has modified them enough so that the restriction enzymes will not recognize them as foreign invaders. A common way of doing this is the addition of a methyl group onto the DNA sequence.


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Name: __Sebastian de Armas_ BIOL123-Fall 2014 Section # __105__

pGLO: Session 2 Post-lab Assignment Due at the start of pGLO: Session 3

1. (1pt) Fill in the following table with the data from your table’s experiments in pGLO: Session 1.

Table Avg # ofglowing cfus

Table Avg # ofnon-glowing cfus

Observations &Notes

“Wt”-pGLO xxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxx-Positive control (10uL) 0 0 There were lots of non

glowing colonies-Positive control (50uL) 0 0 There were tons of non

glowing colonies-Negative control (10uL) 0 0 No colonies grew-Negative control (50uL) 0 0 Only a few colonies grew,

they didn't glow-Experimental Plate (10uL) 746 0 All the colonies are

glowing-Experimental Plate (50uL) 6804 0 All the colonies are

glowing“M”-pGLO xxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxx-Positive control (10uL) 544 0 All the colonies are

glowing-Positive control (50uL) 2000 0 All the colonies are

glowing-Negative control (10uL) 0 0 No colonies grew, they all

died-Negative control (50uL) 0 0 No colonies grew, they all

died-Experimental Plate (10uL) 42 10 Some were glowing,

some were not-Experimental Plate (50uL) 64 18 Some were glowing,

some were not

2. (1pt) Do the results make sense for the positive control plates? Why or why not?

Yes the results make sense because on the wild type we see growth and not glowing as the araC and GFP genes are left in tact and there is a lack of arabinose and antibiotic meaning that the number of colonies will grow. The results also make sense on the mutant type because in the presence of


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arabinose and the insertion of a transposon into either the araC or GFP genes we will see some glow and some not glow.

3. (1pt) Do the results make sense for the negative control plates? Why or why not?

The results do make sense for both the wild and mutant types as for the wild types they are sensitive to kanamycin and therefore will die (producing zero of everything), while the mutant types are resistant to kanamycin and ampicillin meaning that it will survive in those conditions and continue to display the results of a transposon insertion on glowing/not glowing.

4. (1pt) Do the results make sense for the experimental plate for the “Wt” pGLO experiment? Why or why not?

The results do make a lot of sense as the araC and GFP genes in the wild type are left in tact, therefore in the presence of arabinose and ampicillin (which they are resistant to) they will grow and glow (the GFP protein will be expressed, leading to glowing) for essentially all the colonies.

5. (1pt) Do the results make sense for the experimental plate for the “M” pGLO experiment? Why or why not?

The results do make sense for the mutant pGLO experiment as some will glow and some will not glow. The reason for this is because the transposon may have inserted itself into either the GFP gene or the araC gene, preventing the GFP gene from being expressed in the presence of arabinose causing some to not glow.


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Restriction Digestion Experiment

6. (0.5pts) Which enzymes would you like to use in your restriction digestion experiment? PciI and BsteII

7. (0.5pts) Does digestion of the pGLO plasmid by these enzymes separate the araC and GFP genes from one another?

Yes, the PciI cuts at 4748 bp and the BsteII cuts at 1077 bp

8. (0.5pts) Do these enzymes work with 100% activity in the same buffer system? Which buffer system will you be using in your experiment?

No, the BsteII has a 50% activity at 37 degrees Celsius . We will be using the NE buffer 3.1

9. (0.5pts) Do these enzymes cut the EZ-Tn5 transposon?

They do not, yay!

10. (0.5pts) Following restriction digestion, are the resulting DNA fragments of different lengths? List the number of fragments produced and their lengths here.

They are different lengths; there are 2 fragments:Fragment 1: length of 3671 base pairs, cutting from 1078-4748 bpFragment 2: length of 1700 base pairs, cutting from 4749-1077 bp

11. (0.5pts) Are any of the DNA fragments less than 200bp apart in size? Why would it be an issue if your experiment produced fragments less than 200bp apart in size?

No, and it would be an issue as when we would run the fragments through the gel electrophoresis the fragments would be too close to be able to tell the differences between them

12. (0.5pts) Do any of your enzymes require the addition of BSA? If so, which ones?

No


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13. (1pt) Draw a timeline for your experiment that includes the order in which enzymes should be added to the reaction and if they require the addition of BSA, the temperatures & lengths for incubation, and the temperatue and lengths for deactivation (if necessary).

Timeline:

At zero minutes we will add the PciI to the solution containing the desired DNA fragments (add them to the DNA at 37 degrees celcius for 1 hour)

At 60 minutes we must raise the heat to 80 degrees celcius for 20 minutes (PcI’s heat inactivation period)

At 80 minutes we must add the BsteII to the solution of DNA at 60 degrees celcius, so we have to lower the temperature from 80.

At 85 minutes we remove the entire solution from incubation as the reaction is complete

24. (0.5pts)How long will it take to complete your experiment?85 minutes


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Chp3 PGLOMutagenesis