Experiment 3: Transposon Mutagenesis

Overview using Vector NTI: In this module you will learn to use an in vitro transposition system to generate a random collection of insertion mutations in pNIG6. The goal will be to isolate a transposon insertion in the nimX insert of pNIG6 and then determine whether the insertion inactivates nimX function. Transposons are mobile DNA elements that can transpose from one DNA sequence into another. They have been studied since before the physical nature of the gene was understood and have been used as tools to create mutations in vivo and in vitro. NEB’s GPS-1 genome priming system supplies a transposon that is a modified version of the bacterial transoson, Tn7. The transposon is delineated by direct repeat sequences, which are recognized by a recombinase enzyme called a transposase. Transposase inserts the repeats and everything inbetween them into random sites on other DNA molecules. The GPS transposon carries a bacterial selectable marker which allows you to detect plasmids containing the transposon by antibiotic resistance. The NEB transpose is a modified transposase enzyme (TnsABC*) that is highly efficient in vitro. Insertion of the transposon into a gene will typically inactivate the gene, allowing one to locate specific genes in large inserts by insertional inactivation. The transposon also carries sequencing primer binding sites at each end, allowing one to use a collection of random transposon insertions to sequence large inserts without having to make subclones.

First you’ll perform in vitro transposition reactions using pGPS2.1, which carries a mini-transposon containing a gene conferring bacterial resistance to chloramphenicol, and pNIG6, which carries a 2.5 kb genomic fragment containing a functional nimX gene. You’ll select clones that contain the mini-transposon integrated in pNIG6 by transforming the reaction mixture into Invitrogen’s DH5 α electrocompetent cells and selecting for resistance to both AMP and CHL. You should study the GPS-1 manual to understand the basis for the selection and why it is impossible to recover pGPS2.1 or pNIG6 alone. Also, consider whether selection for CHL resistance alone would have been sufficient for our purposes. Be prepared to set up the transposition reactions THE SECOND YOU WALK IN THE LAB – this means you should have your recipe written out in advance, which in turn means that you will have done your homework ahead of time. You should also consider how the restriction digestions in the protocol below can be used 1) to differentiate between transposon hops into the pNIG6 vector sequences versus the nimX genomic region sequences, and 2) map the approximate site of the transposon insertion. The latter is a topic in your homework assignment. NEGATIVE participation points will be given out to all members of any group that is not ready to do the double digests when you reach step 7 below.

Chronological Protocol:

1. Set up and run your GPS-1 Transposition Reaction according the manual. The TAs will provide you with 80 ng/ul stock of pNIG6 DNA, 10X GPS Buffer, 0.02 ug/ul pGPS2-1, ddH2O, Start Solution, an empty culture tube, either NZY+ or SOC liquid medium, 3 L+AMP plates and 3 L+AMP+CHL plates.

a) Mix the following reagents (per 20 μl reaction):

2 μl 10X GPS Buffer (Reagent 1)

1 μl pGPS1.1 or pGPS2.1 Donor DNA (0.02 μg) (Reagent 2a or 2b)

1 ul of pNIG6 DNA

14 ul ddH2O

18 μl Total Volume

Mix well by pipetting up and down a few times.

b) Add 1 μl TnsABC* Transposase (Reagent 3) to each tube. Mix again.

c) Incubate 10 minutes at 37°C (30°C for BAC targets). This is the assembly reaction.

d) Add 1 μl Start Solution (Reagent 4) to each tube. Mix well by pipetting up and down a few times.

e) Incubate 1 hour 37°C (30°C for BAC targets). This is the strand transfer reaction.

f) Heat inactivate at 75°C for 10 minutes. Note: 65°C is not adequate.

2. Transform DH5α electrocompetent cells with your GPS reaction

a) Dilute 2 ul of the reaction with 18 ul sterile, ddH2O and ask the TAs for a tube of DH5α electrocompetent cells. Keep the diluted reaction and the cells on ice.

b) Add 2 ul of the diluted reaction to the cells and mix by pipetting.

c) Take your ice bucket and P-20 pipettor to the TAs by the electroporation unit in the back corner of the lab. The TAs will have a chilled electroporation chamber ready for you and will walk you through the process.

d) Add 1 ml of NZY+ or SOC medium to the electroporation chamber to collect all the cells and transfer the cells + medium to a culture tube provided by the TAs. Incubate the transformation mixture at 37 degrees shaking for 1 hour.

e) While the transformation mixture is incubating, label the L+AMP and L+AMP+CHL plates as follows:

L+AMP 1:100 dilution (your group number)

L+AMP 1:10 dilution (your group number)

L+AMP undiluted (your group number)

L+AMP+CHL 1:10 dilution (your group number)

L+AMP+CHL undiluted (your group number)

L+AMP+CHL remainder (your group number)

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3. Make 1:10 (100 ul plus 900 ul medium) and 1:100 (10 ul plus 990 ul medium) dilutions of the transformation mixture using NZY+ or SOC medium. Plate 100 ul of the undiluted, 1:10 dilution, and 1:100 dilution of the transformation mixture on the appropriately labeled L+AMP plates. Plate 100 ul of the 1:10 dilution, the undiluted transformation mixture, and the remainder of the transformation mixture on the appropriate labelled L+AMP+CHL plates. To plate “the remainder” of the transformation mixture, spin the remaining transformation mixture for 20 seconds in the microfuge, remove all but the last ~ 100 ul of medium, resuspend the pellet in the remaining medium by vortexing, and plate all of that on one plate.

4. Miniprep 6 of your AMP+CHL resistant colonies using the Qiagen kit.

a) Transfer ~1.5 mls of each cell culture into labeled, 1.5 ml centrifuge tubes.

b) Harvest the bacterial cells by centrifugation in a the microcentrifuge at full speed for 1 min at room temperature (15–25°C).

c) 1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 containing RNase.

d) Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

e) Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times and vigorous shaking. The solution should become cloudy.

f) Centrifuge for 10 min at full speed in a table-top microcentrifuge. A compact white pellet will form.

g) Apply the supernatants from step 4 to the QIAprep spin column by pipetting. Label the tops of the spin columns.

h) Centrifuge for 30–60 s at full speed. Discard the flow-through.

i) Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s at high speed.

j) Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the

flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

h) Place the QIAprep column in a clean 1.5 ml microcentrifuge tubes that have the lid cut off (use scissors). To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min at high speed.

j) Transfer the DNA solution to a clean, labeled, 1.5 ml microcentrifuge tube and store on ice. NOTE: there is no EDTA in EB, so keep the DNA cold to minimize the effects of any contaminating nucleases.

5. Digest the minipreps and 0.2 ug of pNIG6 (use 0.2 ug/ul stock) with XbaI plus PstI (NEB buffer 3 plus BSA), run the digests on a gel, and determine whether each clone has a Tn hop in the vector or in the nimX genomic insert. Use Vector NTI and your GPS manual to help you interpret your results. You’ll be given 280 ul of a master mix containing buffer and enzyme. Transfer 35 ul of master mix to each of 7 1.5 ml tubes. Add 5 ul of each miniprep DNA to tubes 1 through 6. Add 1 ul of pNIG6 and 4 ul ddH2O to tube 7. Incubate at 37 degrees for 1 hour.

6. Add 4 ul of 10X loading buffer to each reaction. Load 15 ul on an agarose gel along with MW markers and run the gel for 30 to 45 minutes. Get a photo of your gel when it’s ready and analyze the results as follows:

a) Which clones have the GPS2.1 transposon hopped into the vector

b) Which clones have the GPS2.1 transposon hopped into the insert.

c) Any clones that don’t look like that predicted for either (a) or (b)?

7. Map the approximate site of the transposon insertion in one of your clones that has a transposon hop into the insert.

a) Do the two, double digests with the enzymes you determined in your homework assignment for this lab module. Use Vector NTI and your GPS manual to help you interpret your results. The TAs will be prepared with reagents necessary to do the double digests (1 hr at 37 degrees).

b) Run digest a gel along with MW markers and record the results for analysis later.

NOTE: Groups not ready to do this when step 6 is complete will get negative participation points.

8. Transform an A. nidulans Y306H strain with 10 ul of one of your clones that contains a Tn hop into the nimX genomic region (the insert of pNIG6). Use the procedure included with the Experiment 2 lab protocol.

9. Patch 20 transformants onto two plates containing medium lacking uracil, incubate one plate at 32 degrees and the other at 43 degrees. See patch plate template provided below. Analyze these results in the next lab period and determine whether your Tn insertion inactivated the nimX gene. We will use the positive and negative control transformants from experiment 2 as controls, which should be patched on each plate.

10. Record and Discuss Results

Determine the total number of AMP-resistant transformants per ng of pNIG6 in your transformation. Determine the total number of AMP AND CHL resistant transformants per ng of pNIG6 in your transformation. What plasmid(s) may be present in AMP-resistant colonies? What about AMP plus CHL resistant colonies? If you had used L+CHL plates for selecting transposon insertions in pNIG6 instead of L+AMP+CHL, would that have affected the types of insertion events you would have recovered? If so, how? If not, why not?

What percentage of pNIG6 plasmids in the GPS reaction were “hit” by a transposon (contained a hop somewhere in the plasmid)? What percentage of the clones you analyzed by restriction digest contained a hop in the insert? If the site at which the transposon inserts is random, what percentage of the clones should have a hop in the insert? What is the approximate location of the transposon insertion in the insert that you mapped by double digest (give the location as a “best guess” nucleotide position in pNIG6 using the Vector NTI numbering system for pNIG6. Was the function of the nimX gene on the plasmid you transformed into Aspergillus inactivated or not? What location(s) in the insert of pNIG6 might you propose could be the site of transposon insertions that would NOT inactivate nimX function?

DNAs:

80 ng/ul pNIG6; 20 ng/ul pGPS2.1

Grid for pathing Aspergillus transformants

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