Team:Wageningen UR/Notebook/Journal/Quorum Wet
Quorum Sensing Wet-lab
By: Natalia Giner Laguarda
Here you can find Natalia's work on the wet-lab experiments of the Quorum Sensing system she made. Curious about her results? Read more about it here.
May
Week 1 (1st of May - 7th of May)Getting started, proposal writing, office decoration.
Week 2 (8th of May - 14th of May)
More proposal writing. Becoming an expert in SnapGene by watching YouTube tutorials!
Week 3 (15th of May – 21st of May)
More Snapgene. Started 3A Assembly method.
Week 4 (22nd of May – 28th of May)
Designed primers, experimental setup and applied for BioBricks.
June
Week 5 (29th of May – 4th of June)Modified experimental setup and made illustrations of it in Illustrator. Subsequently became an expert in Illustrator by watching YouTube tutorials!
Week 6 (5th of June – 11th of June)
Finalized construct design.
Week 7 (12th of June – 18th of June)
Finally ordered primers and BioBricks.
Week 8 (19th of June – 25th of June)
Prepared media, made E. coli electrocompetent cells and prepared some agar plates.
July
Week 9 (26th of June – 2nd of July)RFP, CRFP, NRFP, lactose promoter + RBS were transformed from iGEM plates and PCR'ed + purified. Streaked plates, made minipreps and glycerol stocks.
Week 10 (3rd of July – 9th of July)
Ligation 1 (lactose promoter+RBS+ RFP) suceeds. Made glycerol stock of it and sent sample for sequence analysis. Ligation 2 and 3 failed. Moreover, CRFP lacks the ATG start codon and has an unexpected frameshift. NRFP lacks the stop codon.
Week 11 (10th of July – 16th of July)
Design of more primers to fix both CRFP and NRFP.
Week 12 (17th of July – 23rd of July)
PCR addition of ATG to CRFP (CRFP2). PCR addition of RBS to CRFP (CRFP3). PCR addition of stop codon to NRFP (NRFP2). PCR'ed COLE7 and T4 Endolysin from their sources.
Week 13 (24th of July – 30th of July)
Ligations 2 and 3 failed 3 times in a row. Succeeded in the 4th try. Transformation, colony PCR, Q5 PCR of the new set of genes needed for experiments 2 and 3:
- T4 Holin from a requested GeneBlock
- LuxI
- LuxR
- RBS+CFP
- RBS+GFP
- Promoter LuxR-AHL
August
Week 14 (31rd of July – 6th of August)Ligation 2, ligation 3, ligation 5, ligation 6 succeeded. Ligation 4, ligation 7, ligation 8, ligation 11 and ligation 10 failed. Repeated ligation 4, 7, 8, 11 and 10. Failed again. Ligation 11 succeeded.
Week 15 (7th of August - 13th of August)
Holiday! - Out of office.
Week 16 (14th of August - 20th of August)
Holiday! - Out of office. Week 17 (21st of August - 27th of August)
Made liquid cultures, which were subsequently miniprepped and colony PCR'ed. Ligations 4, 7, 8, 10 and 11 were sent for sequencing. None of them worked as all of them were empty, starting to doubt the 3A assembly method. Repeated the whole process, but to no avail. Moved to Golden Gate assembly: everything suddenly worked on my first try. Made a change in the Quorum Sensing approach, ligation 7 and 9 are scratched from now on onwards, I'm going to take a new approach:
- Fluorophore-only ligation (GFP) ---> Acts as negative control
- QS ligation (GFP + LuxR + aiiA under pLac) ---> Acts as negative control
- Reporter QS (GFP + Lux I + LuxR)
- QS ligation 2 (GFP + Lux I + LuxR + aiiA under pLac)
Week 18 (28th of August - 3rd of September)
New set of PCRs in order to add Golden Gate overhangs to everything:
- pLac vector
- NRFP2 for ligation 2
- NRFP2 for ligation 4
- CRFP3
- pBAD vector
- COLE7
- T4 Endolysin
- T4 holin for ligation 7
- T4 holin for ligation 8
- Reporter QS
- aiiA
Week 19 (4th of September - 10th of September)
All the PCR products are ready and purified, but the backbones refuse to be amplified. Stamatios gave me a pair of primers annealing in the resistance of the backbone. Therefore, I moved on to a new Golden Gate assembly method in which each ligation was going to have 1 extra part (as a result of the amplification of each backbone in two parts). Both parts of both vectors (pBAD and pLac) were succesfully amplified. I started the Quorum Sensing ligation and picked colonies until I found a good one. QS ligation (without LuxI) was never decided to be done, but LuxI contains around 100 base pairs and I never realized this while assessing the colonies (colony PCR, restriction analysis, spreading plates) that luxI was not there until I sent my 5 samples for sequencing. The reason for sending 5 samples is that QS ligation is extremelly long and hence 5 primers were needed to get the whole sequence. Everything was perfect but luxI was not there, which saddened me. Then I was happy, because this unexpectedly became the best negative control ever. I started the rest of ligations as well:
- Ligation 2 (amino)
- Ligation 4 (carboxi and amino)
- Ligation 5 (cole7)
- Ligation 7 (endolysin + holin)
- Ligation 8 (holin)
- Ligation 2 (amino)
- Ligation 4 (carboxi and amino)
- Ligation 5 (cole7)
- Ligation 7 (endolysin + holin)
- Ligation 8 (holin)
Week 19 (11th of September - 17th of September)
Only reporter ligation was grown from a glycerol stock and made the QS reporter as well. Repeated Ligation 7 and picked all the possible colonies, miniprepped and analyzed them through restriction digestion and = of course I used colony PCR as well. Finally i found a colony which achieved to insert the gene. But of course, it has a mutation in the gene. It was not possible to pick any perfect colony. The conclusion was that the mechanism was that lytic that there was no possibility that bacteria was contianing the perfect double gene mechanism as it was to lytic to the cells.
Week 20 (18th of September - 24th of September)
Transformed the QS ligation and amplified pSB4K5 with the Golden Gate overhangs. Started experiment 1 (Bimolecular fluorescent recombination of RFP), it completely failed. This experiment was run with the lysis protocol of JosE. Miniprepped 8 colonies of Ligation 7 and analyzed them trough restriction digestion. Sent one for sequencing as well, but it was once again empty. Based on the fact that this super lytic ligation was done millions of times and all the possible colonies were tested, it was concluded that the system was lytic to such a degree that it would never be able to be expressed by a single bacterium. Lytic mechanisms… still working, i have them all, for Ligation 7 I have one successful but it has one mutation, which should not matter. I ran the experiment of the lytic mechanisms in pSB1C3 by inducing with L-arabinose. Results look good so far:
- COLE 7 works perfect
- T4 holin works perferct
- T4 endolysin gives a lot of noise
- Ligation 7 works, but not 100%
October
Week 21 (25th of September - 1st of October)QS experiment done. Bimolecular fluorescent recombination experiment was repeated as well, = but with two different approaches: Sonication rather than lysis protocol Biological lysis rather than lysis protocol (for this I cotransformed all the strains with my COLE7 system), checked on gel.
Week 22 (2nd of October - 8th of October)
Decided to tackle 2 new approaches:
- Quenching GFP by REACH2
- Quenching GFP by the M2 fragment from influenza
Week 23 (9th of October - 15th of October)
Started all the 11 ligations in parallel, from transformation to cPCR and sequencing. Repeated failed sequences (GFP cleavable quencher, TEV protease (3 times), GFP quenched). I got: QS in 4K5, L6 IN 4K5, aiiA in 2C3, l6 fixed, l7 fixed. And repeated oligo annealing with a new protocol as well. Made the Zymo Mix&Go competent cells to see whether this improves my quality of life.
Week 24 (16th of October - 22th of October)
Still couldn't get the Quenchd GFP assembly to work... Repeated it once again with new electrocompetent cells, transfomed in duplo and with different concentrations. Again empty plate, but after some more time in the incubator, something grew. I got the GFP cleavable quencher and GFP M2 absorbed cleavable, but one was mutated and the other one empty. Provided that this was the 4th time i was trying those and they still didn't work, I decided to move forward with the project, as time becomes pressing. Made liquid cultures for:
- QS experiment (with inducible system)
- 8 lytic mechanisms in both backbones
- Quenching experiment
