Matt Gethers/CRI, Thailand/Project Summary/HmgR and HmgA Mutant Generation

=HmgR and HmgA Mutant Generation=

HmgR Summary:
 * I putatively cloned the entire knockout cassette (HmgR upstream and downstream fragments with Lox/Gentamicin - pKn006) into DH5&alpha; - still needs to be sequenced.

HmgA Summary:
 * I've cloned the HmgA upstream and downstream fragments into pKn003, but have been unable to get the Lox/Gentamicin cassette into this construct.

I received this project with most of the components in tact. p'So had digested pUC18 both with SmaI and PstI (separate digests) and had amplified the HmgA upstream fragment (designated 2), the HmgR upstream fragment (3), and the HmgR downstream fragement (4). All that was left was to amplify the HmgA downstream fragment (6.16.08 and 6.17.08). After amplifying, I digested with NcoI (6.17.08), blunt-ended (6.18.08), and ligated into SmaI-digested and blunted pUC18 (6.18.08) to form pKn001.L1. I simultaneously ligated the HmgR upstream fragment into PstI digested pUC18 (the fragment was already digested) to form pKn002.L1. I transformed these into DH5&alpha; (6.20.08) and did a blue-white selection to screen colonies initially.

I selected what appeared to be white colonies from each transformation and streaked out on fresh plates. From these, I found that the pKn001 had yielded several white colonies and colony PCRs (6.25.08) confirmed the presence and direction of the construct. I made two glycerols and preps pKn001.P1 and P2 (see these links for sequence information). pKn002 failed to yield any white colonies, so I religated the HmgR upstream fragment into pUC18/PstI to form pKn002.L2. I transformed this into DH5&alpha; (6.27.08) and got several white colonies. I streaked these colonies out on separate plates and selected 7 promising candidates to do colony PCRs (7.2.08). The PCRs confirmed the placement and directionality of the HmgR upstream fragment. I made two glycerols and preps - pKn002.P1 and P2.

I digested pKn001.P1 with HincII (7.4.08) and pKn002.P1 with SphI and HindIII (7.4.08). I blunt-ended the pKn001.P1 digest (7.4.08) and gel purified the pKn002.P1 digest. I ligated the HmgA upstream fragment into pKn001.P1 to produce pKn003.L1 and ligated the HmgR downstream fragment into pKn002.P1 to produce pKn004.L1. I transformed both of these into DH5&alpha; (7.8.08), but colony PCRs for both pKn003 and pKn004 were negative. Important to note that I should have gotten nothing for a failed cloning step in the pKn003 PCR, but I got a small product about the size of the failed (and expected) pKn004 product.

As both cloning steps failed, pKn003 due to adding the wrong parent vector (I think) and pKn004 due to poor purification, I did the digests over again. I digested pKn002 with SphI-HindIII again (7.10.08) and ran on a PCR clean up column to get rid of the 10 bp fragment rather than running out on a gel. I also digested pKn001 with HincII (7.10.08) and used the End-It kit on the product (7.10.08). I did the ligations on Monday 7.14.08 to form pKn003.L2 and pKn004.L2, then transformed into DH5&alpha; (7.14.08). After two rounds of colony PCRs (7.15.08 and 7.21.08), I found two colonies containing the correct pKn003 and made glycerols and preps: pKn003.P1 and P2. The pKn004 transformation failed again, however. I digested pKn002 with SphI-HindIII again (7.21.08), this time I digested serially and with some controls included. I also took some digested vector and used phosphotase (7.22.08) to reduce my background. I ligated HmgR downstream fragment into both the vector treated with phosphotase and vector not treated with phosphotase to form pKn004.L3 and L4 respectively. I transformed pKn004.L3 and pKn004.L4 into DH5&alpha; (7.23.08) and after two rounds of screening with colony PCRs (7.24 and 7.25.08), I found two colonies, made glycerols and preps, pKn004.P1 and P2.

I digested pKn003.P1 with BamHI, pKn004.P1 with SphI, and pCM351 (which has the Lox/Gent cassette) with SacI and NdeI. I blunted everything and ligated to form pKn005.L1 and pKn006.L1 respectively. Long story short, I ended up with two clones for pKn006 and nothing for pKn005.