Matt Gethers/CRI, Thailand/Project Summary/HmgR Purification and Binding Analysis

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HmgR Purification and Binding Analysis

Summary:

  • I have purified HmgR* and failed to get a clone of wild-type HmgR. In my "HmgR Isolation Box" in the -20, I still have digested HmgR ORF and digested pET-11a should someone want to repeat the cloning. All protocols for generating the plasmids I've presented above can be accessed from their wiki pages.
  • Regarding the cloning of the wild-type HmgR, the interesting thing from the diagnostic digest is that PstI has no effect on the prep take from cultures grown throughout the day. Even if the HmgR ORF is missing, there is a PstI site on pET-11a, so I would expect the vector to be linearized, but I saw no evidence of linearization in the 4 different samples I prepared. I'm not sure what's going on.
  • I performed the binding assay with both HmgR* and GpxR, the former yielding some encouraging results while the latter's performance is not inconsistent with previous experiments.

After amplifying HmgR ORF (6.18.08) with PFU from Viventis, I ligated the ORF into pET-11a to form pIs001.L1 and transformed (6.20.08) into DH5α. To ensure that I got a single colony, I selected one of the transformants and streaked it out on an LB amp plate and then selected two colonies to prep and make glycerols. The prep from both of these are pIs001.P1 (I gave them the same name because they should be from the same clone).

I transformed pIs001.P1 into BL21 DE3 (6.27.08) and did a mini isolation protocol (7.2.08) to determine if I managed to get induction. After verifying the expression of an ~30 kDa protein (consistent with HmgR), I did a larger isolation protocol (7.4.08) and then purified the product both on a heparin column (7.11.08) and then on a Q-sepharose column (7.14.08).

It was after reviewing the sequencing of pIs001.P1, I identified a single base insertion and realized that it's placement would result in a protein (I've named HmgR*) nearly indistinguishable from wild-type HmgR. Reviewing the sequencing from a second clone (6.27.08 transformation) transformed with pIs001.L2 showed other abnormalities. We decided to repeat the cloning using a better high fidelity polymerase.

After amplifying the HmgR ORF with HotStar polymerase (7.23.08), I ligated the ORF into pET-11a to form pIs001.L3 and transformed (7.24.08) into DH5α. After prepping two colonies pIs001.P5 and P6, I transformed first into BL21 (7.26.08), then into BL21 DE3 (7.30.08). In both cases, I failed to get induction (7.29, 7.31, 8.1.08). We theorized that the digested pET-11a (6.17.08) I used for the ligation could have suffered damage to the promoter, so I digested fresh pET-11a (8.2 and 8.5.08) and repeated the ligation of HotStar ORF into pET-11a to form pIs001.L4. I transformed (8.2.08 and 8.5.08) this into DH5α (note I also used old vector to see if there's a difference), but had difficulty getting colonies. After two more attempts at cloning with fresh pET-11a, I successfully transformed pIs001.L8 into DH5α (8.6.08). I prepped two colonies to form pIs001.P7 and P8 and transformed these into BL21 DE3 (8.8.08). I still failed to get induction (8.9.08), however.

At this point, I reviewed the sequencing of pIs001.P5 and pIs001.P6 and found that neither contained the HmgR ORF. After doing a PCR (8.11.08) on P7 and P8, I saw that they, too, were missing the HmgR ORF (or it had at least mutated). This was particularly odd as the colony PCR (8.6.08) taken of the same colonies prior to growing up a culture showed a product at the correct length. Mayuree thought I had a mixed population in the colony, so I transformed pIs001.L8 into DH5α (8.11.08) once more and did two things. First, I selected colonies and did colony PCRs (8.12.08) to confirm the ORF was present immediately after transformation. While the products were a little faint, this held true. With the colony suspension, I streaked out a plate to hopefully select a single homogeneous colony. I also grew up cultures both with and without extra glucose and amp to see if leaky expression was causing a strong negative selection against ORF carrying cells ((see notes from 8.12.08). A diagnostic digest done on the preps of these cultures and a positive control suggested this theory was false, though.

I did perform the binding assay both with GpxR and HmgR*. While GpxR showed very weak affinity for both gpx1 and hmgA promoters, HmgR* yielded some promising results for both promoters.

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