Team:Wageningen UR/Notebook/Alba
Molecular Timer Kill Switch
By: Alba Balletbó Canals
In the notebook of Alba you can read all about her time building a kill switch based on a molecular timer. Click here to go the results page.
May
Week 1 (13th of May – 19th of May)
We got our own office and started working full time. Divide all the subprojects among the team members.
Week 2 (20th of May – 26th of May)
Research and literature study.
Week 3 (27th of May – 2nd of June)
Research and literature study.
June
Week 4 (3rd of June – 9th of June)
Research and literature study.
Week 5 (10th of June – 16th of June)
Writing the proposal and introduction presentation.
Week 6 (17th of June – 23rd of June)
Writing the proposal and introduction presentation.
Week 7 (24th of June – 30th of June)
Finish writing the proposal. Working on the design of the synthetic circuit of the circadian oscillator.
July
Week 8 (1st of July – 7th of July)
Design and ordering of primers for the circadian oscillator. Amplification of the genes of interest* coming from a genomic DNA of Synechococcus elongatus (Strain PCC 7942). *Genes of interest from Synechococcus elongatus (Strain PCC 7942): kaiA, kaiB, kaiC, rpaA, rpaB and sasA.
Week 9 (8th of July – 14th of July)
Successfully amplified all genes of interest except for kaiC. Ligation into the plasmid backbone (pSB1C3) and transformation into Escherichia coli chemically competent cells of the successfully amplified genes. Assistance to the Annual Congress of Biotechnology (BAC) in Madrid.
Week 10 (15th of July – 21st of July)
Vacation week.
Week 11 (22nd of July – 28th of July)
Repeat amplification, ligation and transformation of the oscillating genes. Successfully amplified, ligated and transformed all the genes of interest.
Week 12 (29th of July – 4th of August)
Grow colonies containing the desired genes. Miniprep and send for sequencing.
August
Week 13 (5th of August – 11th of August)
Check sequencing results. Transformation failed for some of the genes. Unexpected gene coming from a different species present. Transfer of genes due to contamination from other members experiments. Ligation and transformation of Plasmid Nº1 (plasmid only containing kaiABC, no promoter yet). Oligo annealing of the kaiBC promoter. Amplification of GFP from a plasmid containing the sequence of the GFP (plasmid provided by one of the supervisors). Ligation of the kaiBC promoter with the GFP into the plasmid backbone (pSB1C3) and transformation into E. coli chemically competent cells (Plasmid Nº3). Miniprep and send for sequencing Plasmid Nº1.
Week 14 (12th of August – 18th of August)
Check sequencing results for Plasmid Nº1. Mismatches and gaps present. Miniprep and send for sequencing a different colony. Grow colonies containing Plasmid Nº3. Amplification of the rhamnose-inducible promoter (rhaP(BAD)), as well as the tetracycline-inducible promoter (pTet). Also, amplification of the kaiABC operon, rpaA, rpaB and sasA with new primers containing RBS and overhangs suitable for ligation with rhaP(BAD) and pTet. Repeat ligation and transformation of Plasmid Nº3. Colony PCR of Plasmid Nº3. Miniprep and send for sequencing different colonies that show to contain Plasmid Nº3. Ligation and transformation of rpaA, rpaB and sasA separately into the plasmid backbone.
Week 15 (19th of August – 25th of August)
Check sequencing results for Plasmid Nº1. Still present the same gap. Check sequencing results for Plasmid Nº3. Mismatches and gaps present. Repeat ligation and transformation of Plasmid Nº3. This time, ligating piece by piece. Grow the colonies congaing the genes rpaA, rpaB and sasA. Miniprep and send for sequencing the plasmids containing rpaA, rpaB and sasA. Right sequencing results for rpaB and sasA. Ligation and transformation of PkaiBC+GFP, rhaP+kaiABC Operon and kaiA Alone, into different plasmid backbones (all pSB1C3). Colony PCR several obtained colonies.
Week 16 (26th of August – 1st of September)
Grow the right colonies that show to contain the desired genes or constructs. Miniprep and send for sequencing. Repeat ligation and transformation of Plasmid Nº3.
September
Week 17 (2nd of September – 8th of September)
Check sequencing results. Successfully transformed Plasmid Nº1 and Plasmid Nº3 into pSB1C3. Right sequencing results for the two of them, expect for the kaiA Alone. Amplification of a pSEVA plasmid backbone (specifically the SEVA # 4 4 -). Ligation and transformation of Plasmid Nº1 into the new pSEVA plasmid backbone. Miniprep and send for sequencing. Repeat amplification and purification of kaiA Alone. Ligation and transformation of rhaP+kaiA Alone (Plasmid Nº1’) into two different plasmid backbones (pSB1C3 and pSEVA 44).
Also, working on the design of the Toxin/Antitoxin system. For that, 2 different plasmids have been designed.
Week 18 (9th of September – 15th of September)
Miniprep and send for sequencing colonies that show to contain the Plasmid Nº1’. Only colonies present in the pSB1C3, no colonies present in the pSEVA 44. Amplification and purification of the genes, rpaA, rpaB and sasA. Repeat ligation and transformation of Plasmid Nº1into pSB1C3 and Plasmid Nº2 into pSB1C3. Miniprep and send for sequencing Plasmid Nº1 only on the pSB1C3 plasmid backbone. Colony PCR for Plasmid Nº2 did not show the right size for any of the colonies.
Also, amplification of the parts constituting the mazF (Toxin) and MazE (Antitoxin), with the designed primers.
Week 19 (16th of September – 22nd of September)
Repeat amplification of mazF and mazE.
Apparently, the rhaP I amplified is not the correct one, meaning that a part of it is missing. This promoter is regulated by two activators, RhaS and RhaR, which are part of one transcription unit, located in the opposite direction of rhaBAD. New primers have been designed and used to amplify the right full promoter sequence. Also, new primers have been designed to amplify the kaiABC operon which would match the new promoter sequence and plasmid backbone. Ligation and transformation of the rhaP(BAD)+kaiABC Operon into pSB1C3 plasmid backbone.
After several attempts trying to amplify, ligate and transform Plasmid Nº2, the one containing rpaA, rpaB and sasA, under the pTet, but just keep failing, the assembly of that plasmid is not pursued anymore.
Week 20 (23rd of September – 29th of September)
Keep failing on the ligation and transformation of Plasmid Nº1. Design of new primers to perform Gibson Assembly. Amplification of parts with the new primers. Performed Gibson Assembly and transformation into two plasmid backbones (pSB1C3 and pSEVA 44).
Ligation and transformation of the Toxin/Antitoxin system as well as the Toxin Alone into pSB1C3 and pSEVA 65 plasmid backbones.
Week 21 (30th of September – 6th of October)
Miniprep and send for sequencing Toxin Alone, as well as the Plasmid Nº1, in both plasmid backbones. All sequencing results correct. Design and set up the experiments on the plate reader.
October
Week 22 (7th of October – 13th of October)
Working on the molecular thing
Week 23 (14th of October – 20th of October)
Working on the molecular thing