Team:Rice/Notebook
Wetlab Timeline
June
4-6: Wetlab bootcamp! We all got trained in basic wetlab techniques
7-31: Lots of research went into designing our project. We met almost everyday to read papers and design constructs
July
9: Began inoculation of borrowed plasmids
11: Acquired the template, which is referred to as pBEST, that we would be using to make a pCON-araC
12: Restriction digests have been failing. We realized today it was due to bad enzymes and loading dye. We ordered new supplies
13: We performed annealing reactions to make some of our parts, including otsA and otsB, iaaM and iaaH and some of the preliminary thermometers
14: Redid the inoculation of the borrowed “part plasmids”
17: Attempted our first Golden Gate of our parts plus the borrowed part plasmids. Later results would show this did not work
22: Redid the anneals, this time doing phosphorylation reaction first. We decided to make an otsBA operon by PCRing off of an E.coli genome. Attempted another Golden Gate to make a pCON-araC. Later results would show this failed
23: Since our constructs need to be arabinose induced to fluoresce green, our plates only had white colonies, we decided to pick a few and digest them to see what they were. We determined that they were all unreacted ampR plasmid. To try to improve our chances of picking the right colonies, we both plated in arabinose and performed a SwaI digest before transforming. The SwaI digest selectively cuts AmpR
24: Transformed SwaI restriction digest of pIG102 into DH108 comp cells and plated on LB-amp plates and LB-amp-ara plates but they did not show any growth when checked on later
25: Retransformed pIG102 and pIG102 SwaI digest. Conducted Golden Gate assembly of linIG102 and pIG101 to make pIG102, transformed them, and plated them on LB-amp plates. We modified our miniprep protocol because we had been getting such low DNA concentrations by lowering the amount of elution buffer
26: We realized our cassettes were wrong after doing a notI restriction digest. We sent our parts in for sequencing to see what was the issue
28: Transformation of pIG005 failed
31: Prepared P. putida comp cells and plated them on LB plates and two PNS plates with varying levels of sucrose. LB plate showed significant growth and the PNS plates + sucrose showed no growth
August
1: Measured fluorescence of P. putida cultures in relation to the emission and excitation of sfgfp. Prepared P. putida electrocompetent cells
5: Amplified Pcon-araC part out of the pBEST plasmid and purified PCR product
6: Attempted Golden Gate to make pIG101 a, b, and c, each with a different replication origin. Later results showed that they failed and it was likely the AmpR plasmid
7: Made plates with PN media under different conditions to grow P. putida: glucose (25 mM), glucose + NaCl, sodium citrate (13 mM), NaCl (1 g/L)
12: Results of growing P. putida on PN plates under different conditions: sodium citrate showed the most growth, but we are going to use glucose since it is cheaper and more reasonable for field applications. Performed a bsaI, salI,and xbaI restriction digest to remake linIG005 since we discovered that the primers bind nonspecifically to other terminators in the template
13-14: Tried to purify araC from pBEST plasmid, but purifications came out badly, and araC continues to give our wetlab team problems
15: Gblock of pcrIG005 came in. Made part assembly of pSPB440 and pcrIG005 and transformed onto LB + Chl plates
16: Poor transformation results with only green colonies visible which is not what we were looking for. Made 3 separate Golden Gate reactions and transformed them into E. coli: pIG101a, pIG102a, pIG102 positive control (without pcrIG005)
17: Restriction digest of pIG101 and pIG102 using notI. Gel results showed that the bands were of unexpected sizes; they were too small to be the cassette and too small to be part plasmids
18: Made a series of positive controls using our advisor’s plasmids to see if there is any toxicity from the combination of araC and RK2 which contained sfgfp, AmpR, either linIG005b or pSPB616 (tonVT), and either RK2 or p15A origin
20: Results from the transformation of positive controls showed that the only plates with the desired green colonies were the plasmids with the p15A origin instead of RK2 like we had been using. We switched to using the p15A origin as the testing constructs for thermometers
21: Golden Gate of multiple versions of the cassette we could use for thermometer testing. The cassettes included KanR, sfgfp, LacI, and either the p15A or pSC101 origin
22: Greater transformation efficiencies from the new constructs
23: 7 part Golden Gates:
(1) pSPB869b, ConLS, Pa1-LacO1, RBS BB0034,mCherry2, VoigtT, ConR2
(2) pSPB869b, ConLS, PL-Lac1, RBS BB0034,sfGFP, VoigtT, ConR2
(3) pSPB870b, ConLS, Pa1-LacO1,RBS B0034, mCherry2, VoigT, ConR2
(4) pSPB870b, ConLS, PL-Lac1, RBS B0034, sfGFP, VoigT, ConR2
25: Transformation results from new constructs (pSPB871-874) of 7 part Golden Gate with backbones from the 23rd and parts 1-6 were good with mostly white colonies
September
1: Golden Gate assembly of thermometers 1, 3, 4, 7 with pSPB875 and pSPB877 purified PCR products
3: Golden Gate assembly of testing cassette for the enzymes using pSPB413, 608, 449, and 450
6: Miniprep of pIG123. Sequencing results of pIG104 failed
10: Trial run for testing RNA thermometers and using the plate reader
12: Annealed, phosphorylated, assembled, and transformed computationally designed thermometers
13: RNAT101 sent for sequencing
14: Began P. putida and A. thaliana control experiments with flood inoculation
19: Thermometers arrived!
20: Transformed and inoculated thermometers
22: Miniprepped all thermometers
23: NotI restriction digest of RNAT 102-123; Sent RNAT 102-123, except 105 for sequencing
25: Prepared kit thermometer parts and RNAT105
28: Made pcrSPB875
29: DNA cleanup of pcrSPB875 and digested with DpnI
30: Transformation of thermometer cassettes and controls
October
1: Inoculated colonies for RNA thermometer testing in LB+Kan and in LB+Kan+1mM magnesium for cultures at different temperatures. Made 0.59 mM IPTG plates and spotted them with 1 uL of culture and incubated at either 25, 30, or 37 degrees Celsius
2: Took macroscope images of IPTG plate spots. Redid colony PCR on RNAT009 and RNAT021 and there were no bands. Obtained E. coli genome and amplified otsBA from it using oIG42 and oIG43, gel purified it, and transformed on Chl plates
3: Performed colony PCR of thermometers
4: Inoculated A. thaliana with P. putida
6: Seeded plates with P. putida to test flood inoculating versus drop inoculating
8: Electroporation transformation of P. putida
11: Prepared thermometers for time course testing and grew P. putida with different amounts of carbenicillin to test for optimum growth
12: RNAT Testing
14: Electroporation transformation of P. putida
16: Inoculated A. thaliana with P. putida
18: Time course for the thermometers with ramping temperatures