Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 7.27.08

=7.27.08=

To Do:
 * Collect pIs001.P4/P5 BL21 Transformants (7.26.08) and begin HmgR Isolation Protocol. Go as far as spinning down the cells, then place them on ice over night.
 * Make two SDS-PAGE Gels to determine efficiency of induction and to identify peak fractions after purification tomorrow.

Summary:

I successfully grew up and froze the cells. They're ready for tomorrow. I also made 3 SDS-Gels and put them in the 4 degree for the time being.

=7.28.08=

To Do:


 * Resuspend cells in low salt buffer
 * Ask Mayuree about supplementing with the protease cocktail and/or DTT
 * Resuspend in how much?
 * Sonicate cells (unless Mayuree wants to try the French Press Again).
 * Spin down the cells. Collect supernatant in one tube (~10 ml) and pellets in another.
 * Do purification on Heparin Column.
 * Run out fractions as well as pre/post induction cells super/pellet on SDS-PAGE Gels.
 * Gel results recorded under purification protocol.
 * Dialyze sample if prepared.
 * Send pIs001.P5 and P6 off for sequencing.

Summary:

I completed the elution and run samples on gels, but they gels are particularly special, so I think I'm going to have to run them again in the morning. I'm holding off on the dialysis until then, though I did make 2 liters of buffer. I sent off the pIs001 clones for sequencing.

=7.29.08=

Judging by the purification results, I think I failed to induce production of HmgR. I'd like to grow up a few separate colonies from the BL21 plates (both transformations), lyse the cells, and run out the products on a gel to see if I'm getting induction anywhere. I'll use colony suspension so I can use the same colony to start overnights if I decide to push ahead.

To Do:


 * Start 4 4 ml cultures of the 7.26.08 BL21 transformants (2 from each clone, will split into -/+ IPTG later) and do the mini isolation protocol.
 * Run the Products out on a gel and look for induction.
 * Check sequencing for pIs001.P1 again
 * That the clone has an insertion and is still the same length is very unlikely. Check my vector NTI file and check for any other insertions/deletions.

Summary:

Got as far as spinning down the cultures after induction. Will sonicate and run out products tomorrow. I started overnights from the colony suspensions of pIs001.P5 1 and pIs001.P6 2 (used in the mini isolation) so I can start a large culture tomorrow if everything looks good. I didn't get a chance to review the Vector NTI file.

=7.30.08=

To Do:
 * Finish mini isolation protocol and run out products on gel.
 * If induction good, start large culture for isolation tomorrow.
 * If induction bad, talk with Mayuree.
 * Check sequencing for pIs001.P1 again
 * That the clone has an insertion and is still the same length is very unlikely. Check my vector NTI file and check for any other insertions/deletions.

Summary:

I see that the induction failed, but I also realized that I transformed into BL21 rather than BL21 DE3, so I would expect expression to be troublesome. I transformed pIs001.P1, P3, P5, and P6 into BL21 DE3. Tomorrow, I will grow up cultures and try the mini isolation protocol again.

=7.31.08=

To Do:


 * Try the mini isolation protocol again using yesterday's transformants.
 * If induction looks good, start overnights for en masse protocol tomorrow.

Summary:

The induction of the new clones (pIs001.P5 and P6) still failed even when the original clone (pIs001.P1) was expressed properly. Mayuree suggested a fresh transformation and inoculating cultures with many different colonies rather than just one. I did a transformation of P5 and P6 into DE3 this evening and I will try the mini isolation protocol again tomorrow.

=8.1.08=

To Do:


 * Try the mini isolation protocol again using fresh transformants and many different colonies.
 * Check sequencing data to see if "insertion" is OK.
 * Go over initial isolation protocol to see if I'm doing anything significantly differently.

Summary:

Finished the mini isolation again - it looks like the induction failed yet again. I went over the sequencing data - the translation from the vector NTI file and the protein sequence from KEGG both match. Still have to figure out what the insertion would do (i.e. give a ~28 kDa protein?). Need to go over original protocol again.

=8.2.08=

Talked with Mayuree - given that a product shows up in the colony PCR for the HmgR ORF (from DH5&alpha;, at least), we think there's a problem with the pET-11a vector. I used the same pET-11a digest (6.17.08) as for the original clone. If the sample were somehow exposed to DNAses or the enzymes weren't completely inactivated, they might have disabled the promoter region of the vector. I still have an aliquot of undigested pET-11a, so I'm going to digest some of that with NdeI and BamHI again and ligate it into freshly amplified and cut HmgR ORF.

To Do:
 * Amplify HmgR ORF using HotStar
 * Digest both pET-11a and HmgR ORF with NdeI and BamHI
 * Ligate the ORF into digested pET-11a, newly and formerly digested.
 * Transform the constructs into DH5&alpha;

Summary:

Completed transformation. Hope to grow up cultures tomorrow, prep, and transform into BL21 DE3 so I can test for induction.