Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 8.11.08

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8.11.08

After reviewing sequencing data from pIs001.P4 and P5 (first attempt at induction with HotStar ORF), I see that the ORF was not present in the prep. That would certainly explain the lack of induction. I suspect that the same has happened with P7 and P8. Mayuree thinks I have a mixed population in the colonies I'm selecting and recommends streaking out transformants and doing a diagnostic digest to be sure I have the ORF. That would be consistent with the colony PCR yielding a positive result and the sequencing giving a negative one. In parallel, I'll also test if expression of the protein during the time I'm growing it up in culture for a glycerol and mini prep is causing the ORF to be mutated or deleted (Mayuree says this is unlikely, but I'd already started the cultures so I'll finish it). For this last experiment to be meaningful, the colony PCR should give a positive result while a PCR of the prep should give a negative result. I started PCRs this morning including pIs001.P7 and P8 (I realize now that I should have included P3 and P4...) and 4 colonies for which I started cultures this morning. The cultures included glucose and more Amp to hopefully stabilize the plasmid (does DH5α even have T7 polymerase?). If the PCR of the P7 and P8 preps are negative (while the colony PCR was positive), then I'm losing the ORF when I grow up cultures and I can determine if the extra amp and glucose helped stabilize the ORF this evening. If not, I'll just abandon this line of inquiry all together and just focus on getting the pure colony.

To Do:

  • Streak out a pIs001.L8 colony (that already had a positive colony PCR result) on an LB amp plate.
    • When the colonies grow up, do PCRs and/or diagnostic digest to determine purity of colonies.
  • Do a PCR for the HmgR ORF on pIs001.P7 and P8 Prep (should have done P3 and P4 as well)
    • See if I get a positive or negative result. Positive - inconclusive. Negative - I've lost the ORF during the culture.
  • Grow up cultures of pIs001.L8 transformants (some for which I've already done colony PCRs, some new ones) using extra Amp and some glucose to stabilize the plasmid.
  • Do PCRs on the preps from the cultures to see if the ORF is still present.
  • Finish script to help manage sequencing data and finish going through data.

My Schedule for the next 3 days:

  • If the P7 and P8 preps give negative results (no product), then I continue both with trying to isolate a single colony from a streaked plate and prepping from cultures grown up in more amp and glucose to see if I managed to stabilize the ORF - Monday afternoon. Monday evening, I start PCRs on the preps and transform into BL21 DE3 anticipating a positive PCR result. Tuesday morning, run out products on gel. If positive result (product present), grow up DE3 en masse (skip mini induction protocol) and get as far as spinning down the cells. At this point, I'll also take a sample and run it out on a Poly-AC to see if I got the induction to work. If it did, purify HmgR on a column Wednesday, If not, drop it and just try to get the pure clone from the plate streaking again.
  • If the P7 and P8 preps give positive results, don't bother with the cultures, and just try to get the single colony by streaking. Monday night, streak the plates. Tuesday morning, select colonies, do PCRs and grow up cultures. Prep on Tuesday evening and try a diagnostic digest to be sure it worked. Also transform into BL21 DE3. Wednesday morning, assuming everything looks good, grow up culture en masse (skip mini isolation protocol) and take samples in the afternoon to run on a gel. If everything looks good, spin down culture - could possibly purify on Thursday or just leave for someone else.

Summary:

I ran the colony PCR and found that the preps looked very weird (~5x the expected length) while none of the colony PCRs worked and the positive control worked as expected. Taken together, I think this confirms my belief that something is happening to the culture as it grows overnight (whether this is the amplification of a pre-existing mutant subpopulation or the generation of a mutant I don't know) and that I shouldn't use the 8.6.08 transformants to streak out a plate as the colonies no longer appear to contain the plasmid. I'm doing a transformation with some of the remaining pIs001.L8 to generate fresh colonies. Tomorrow, I will select colonies, do colony PCRs, grow up cultures (some in extra amp and glucose, others in normal conditions), and at the end of the day do either a diagnostic digest or PCR on the Prep. I can also use the colony suspension to streak out a plate and then test the colonies Wednesday.

8.12.08

To Do:

  • Run Colony PCRs on yesterday's transformants.
    • I'm hoping for the same result as last time - that all the PCRs show an ~800 bp product. If not, I should abandon my experiment with cultures (below). I also need to streak out a plate to get single colonies assuming this initial PCR works.
  • Grow up two 3 ml cultures for each of 8 colonies - one with 1/1000 Amp, and one with 1/500 Amp and 0.4% glucose.
  • Design a diagnostic digest for the correctly placed HmgR ORF in pET-11a.
  • Assuming the initial colony PCR gives positive results, make glycerols and prep 2 of the colonies (4 tubes total). Do a diagnostic digest to determine if the ORF is present and in the correct location. I'll need to use some pIs001.P1 or P2 prep as a positive control.

Summary:

Started 16 cultures at 0920 this morning. Two master mixes of 25 ml each. To the first I added 25 μl Amp and aliquotted 3 ml to each of 8 tubes. To the second master mix I added 50 μl amp and 500 μl 20% Glucose, then aliquotted 3 ml to each of 8 tubes. Added 10 μl of each of 8 colony suspensions (1 colony in 50 μl water) to one tube of each type. Collected this evening at 2130. Given the Colony PCR result, I selected cultures 7 and 8 to mini prep. After prepping, I digested each sample and pIs001.P1 with PstI and ran out on a gel. Unfortunately, it looks as though the ORF is missing. No Go.

I also streaked a plate of colony 8 of the 8.11.08 transformation - maybe I'll be able to isolate a single colony that has the ORF.

8.13.08

The digest shows that the preps from last night don't contain the HmgR ORF. I streaked out a plate with a colony last night, hopefully I'll get some colonies I can select to PCR later (though I may not do this step).

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