Lab Notebook

---

Week 1
May 5th - May 10th, 2014

Let's get the show on the road!

Starting on a high note, we first transformed the MH1E.coli with pBAD26-PmrA/PmrB, pBAD26-PmrA/PmrB-MUT, pBAD26-PmrC-GFP and pBAD26/CheZ plasmids and selected for mutants resistant to ampicillin. We also streaked the selected colonies. We followed up with transformations using the pDriveKan(m12), pDriveKan(m6), and ΔFos/pSB1C plasmids. Unfortunately, the latter transformations failed which compelled us to repeat the procedure. Inoculated the ΔFos/pSB1C plasmid-carrying bacteria with fresh LB, all done with thought of procuring a nice supply of the pSB1C plasmid backbone, which we indeed succeeded in miniprepping at the end of the week.

Week 2
May 12th - May 17th, 2014

We started off by digesting the freshly procured pSB1C/ΔFos plasmid with pairs of all four BioBrick-compatible endonucleases: EcoRI, PstI, SpeI, Xbal and subsequently put them on electrophoresis to screen for possible mutations. Afterwards, pDriveKan/PmrC–GFP and pJBAD26/CheZ plasmid-carrying bacteria were inoculated for an overnighter, which we later miniprepped for future uses. Towards the end of the week, we transformed bacteria with ligations: m12, m6 and repeated streaking of E.coli/ pDriveKan(m6) and pDriveKan(m12).

Week 3
May 19th - May 24th, 2014

We digested plasmids carrying the m6 and m12 inserts with EcoRI and PstI. Subsequently, we electrophoresised these with PstI, SpeI, Xbal, EcoRI. Later, we repeated the same procedures for the pSB1C-backboned plasmids carrying the BBa_0030 BioBrick part with both EcoRI/PstI and Xbal/SpeI. Screening for mutations returned none.

  Week 4
May 26th - May 30th, 2014

We transformed our bacteria E.coli MH1 with plasmids pSB1C carrying BBa_J04450 part and electrophoresized linear plasmids from the 2013 Distribution – pSB1C, pSB1A3, pS2K3 to replenish our stocks.

Week 5
June 5th - June 8th, 2014

At Polish universities, June usually means exams so we had little time for labwork, yet still: We transformed our dear E.coli with ligations we produced during the last week of work in May. Then, we phosphorylated linear pSB1A3, pSB1C3, pSB1T3 and pSB1K3 vectors, ligated them with PmrA/PmrB-MUT as well as PmrC-GFP and transformed these vectors into E.coli. Next we transformed bacteria ligated with plasmids pSB1C3. Then we inoculated the night bacteria with pSB1C3 carrying both PmrA/PmrB-MUT and PmrC-GFP, isolated and digested these vectors with EcoRI/PstI, and electrophoresized them for screening. Screening came out positive so it's all good... for now.

Week 6
June 30 – July 4, 2014

The winds were favourable to our team members: all exams passed and restocked with new energy, the team is back in the lab. We started off with PCR-ing the PmrA/PmrB(WT) genes as a single construct, as well as PmrA and PmrB separately. Us following up with more PCR, PmrA, PmrA/PmrB (N – term), PmrB and PmrB ( C – term) met with the same fate. We enjoyed it so much that we decided to repeat it, as some technical difficulties occured that rendered the first run fruitless. As our supply of pSB1C3 was running out, we inoculated pSB1C3-carrying bacteria for an overnighter, following with a miniprep, digestion using PstI/EcoRI, then electrophoresis and gel isolation. Finally, ligations comprising the chloramphenicol resistance-carrying plasmid and PCR-ed genes after digestion with the abovementioned enzymes were transformed into our E.coli. As a finishing touch, we transformed bacteria with the transitive pBAD plasmids carrying PmrA/PmrB(WT) and PmrA/PmrB(MUT) obtained after ligations. However, for reasons unknown, the pSB1C3 ligations failed.

Week 10
July 14 – 19, 2014

First we selected for transformants within last week's transformations (i.e. the pBAD and pSB1C3 carrying the PCR-ed genes) and inoculated overnight colonies with LB. Parallely, some of our pDriveKan plasmid stock was digested using EcoRI, electrophoresized and isolated from gel. Subsequently, the PCR products – PmrA/PmrB(WT), PmrA/PmrB(MUT), PmrA, PmrA/PmrB (N-term.) were ligated with the digested pDriveKan and transformed into chemocompetent MH1 E.coli. The next day we miniprepped the pDriveKan plasmid DNA and digested it using PstI/EcoRI, electrophoresised and purified the inserts by gel isolation. Finally, we ligated the inserts with pSB1C3, and transformed them into bacteria. Finally, we PCRed the PmrB, PmrB (C – term), PmrB (N-term) fragments and prepared some more chemocompetent bacteria while at it.

Week 11
July 21 – 26, 2014

Starting the beautiful week, we digested the following inserts on pDriveKan with EcoRI/PstI: PmrA, PmrB, PmrA/PmrB (N –term), PmrB (N – term), PmrB (C– term) as well as pSB1C3/sfYFP, put them on gel to screen for potential restriction site-forming mutations, dephosphorylated the digested plasmids with AP, and isolated pSB1C3 from gel. Following up, we again ligated linear pSB1C3 with product PCR of the former week. And again, transformed the ligations into our bacteria, hoping for the best.

Week 12
July 28 -31, 2014

We performed some transformations with the pDriveKan/PmrA/PmrB(MUT) plasmid. Then, after an overnighter on agar+kanamycin plates, we inoculated cultures carrying PmrA/PmrB, PmrB, PmrA/PmrB (N – term), PmrB (N – term) and PmrB (C – term)-containing plasmids and digested an ample amount of pSB1C3/sfYFP and put it on gel, to isolate the backbone.

Week 15
August 11-14, 2014

We're having ample problems with our procedures, especially with transformations, which seem to yield transformants carrying little to no DNA of interest. We quickly did ligation of PmrA/PmrB(MUT) carrying the BBa_B1006 terminator and PmrC–GFP carrying the same BBa_B1006 to no avail, however. Also, we wanted to insert PmrA/PmrB(MUT) with BBa_B1006 into the BBa_K081005 promoter but encountered similar DNA isolation problems.

We did this until the beginning of September.

The work over summer was hard, not all experiments succeeded (at least, to the extent we wanted them to). But most of them came out positive and so we would like to share with you some of our favourite memories, here in the form of our best gel camera images:

1. pSB1C3/BBa_K081005 2. pDriveKan/PmrA (1) 3. pDriveKan/PmrA (2) 4. pDriveKan/PmrB (1) 5. pDriveKan/PmrB (2) 6. pDriveKan/PmrB (N-term.;1) 7. pDriveKan/PmrB (N-term.;2) 8. pDriveKan/PmrB (C-term.;1) 9. pDriveKan/PmrB (C-term.;2) 10. pDriveKan/PmrA-PmrB (N-term.;1) 11. pDriveKan/PmrA-PmrB (N-term.;2) 12. pSB1C3/PmrA-PmrB(mut)+BBa_B1006	1. pDriveKan/PmrA (1) 2. pDriveKan/PmrA (2) 3. pDriveKan/PmrB (1) 4. pDriveKan/PmrB (2) 5. pDriveKan/PmrB (C-term.;1) 6. pDriveKan/PmrB (C-term.;2) 7. pDriveKan/PmrB (N-term.;1) 9. pDriveKan/PmrB (N-term.;2) 10. pDriveKan/PmrA-PmrB (N-term.;1) 11. pSB1C3/BBa_BK009	1. pDriveKan/PmrB (1) 2. pDriveKan/PmrB (N-term.) 3. pDriveKan/PmrA (1) 4. pDriveKan/PmrA-PmrB(mut) 5. pDriveKan/PmrB (2) 6. pDriveKan/PmrA-PmrB(N-term.) 7. pDriveKan/PmrA (2) 8. pDriveKan/PmrB (C-term;1) 9. pDriveKan/PmrB (N-term.) 10. pDriveKan/PmrB (C-term;2) 11. pDriveKan/PmrA-PmrB(N-term.) 12. pSB1C3

1. pDriveKan PmrA-PmrB (wt)(1) 2. pDriveKan PmrA-PmrB (wt)(2) 3. pDriveKan PmrA-PmrB (mut1) 4. pDriveKan PmrA-PmrB (mut2) 5. pDriveKan PmrA-PmrB (mut3) 6. pSB1C3-PmrA	komentarz - 5 zdjęcie

Retrieved from "http://2014.igem.org/Team:Warsaw/Notebook"