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Team:Groningen/Template/MODULE/Notebook/Secretion
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Notebook
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Secretion
July 21 - July 25
In silico preparation of primers for the Gibson assembly between signal sequence USP45 and modified version of aiiA (BBa_C0060).
In silico production of synthetic gene ssUSP45DspB (BBa_K1365111)
July 23 - August 3
Primer design for the synthetic gene
August 6 - August 8
Obtaining all the BioBricks needed for the secretion system to destroy P. aeruginosa and S. aureus
Performing three transformation to E. coli, one with promoter P2 (BBa_I746104), one with lasI (BBa_K081009), and one with aiiA (BBa_C0060)
The tranformated E. coli was divided in several concentrations over LB agar plates with Chloramphenicol
Three glycerol stocks of E. coli DH5α containing the RBS (BBa_ B0034), the double terminator (BBa_ B0015) and the promoter LasR (BBa_ R0079), were inoculated in LB medium, and grown overnight
The plasmids were isolated out of the overnight cultures, and checked on gel
August 11 - August 15
Assembling the promoters P2 (BBa_I746103) and pLas (BBa_K091117) with RBS and the gene coding for GFP (BBa_ E0040) with double terminator (BBa_ B0015)
The Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled using three-A assembly, the constructs were ligated, and transformed to E. coli DH5α
The transformations were plated on LB agar with kanamycin
All the colourless colonies were plated to fresh plates again, and grown overnight
The construct was checked by colony PCR, ending up with the following constructs: Ps with RBS, pLas with RBS, and the gene coding for GFP with double terminator
Another colony PCR was performed with Phusion polymerase this time, in order to produce a more clean product for a second assembly
The primers and the synthetic gene ssUSP45DspB (BBa_K1365111) arrived
The mrfp insert was cut out of pSB1C3, run on gel, and the linearized vector was purified out of the gel
ssUSP45DspB was ligated with pSB1C3, and transformed to E. coli DH5α
Few colonies were visible after growing the transformation mixture of LB agar plates with chloramphenicol
After digestion, the constructs of the few colonies were checked on gel, only one colony showed the right size for the insert
August 25 - August 29
Obtaining constructs with different promoters and the gene coding for GFP for promoter analysis in L. lactis
An overnight culture of E. coli DH5α with the P2 with RBS construct, pLas with RBS construct, and the gene coding for GFP with the double terminator construct was made
The constructs P2 with RBS, pLas with RBS, the gene coding for GFP with the double terminator and ssUSP45DspB were checked on gel, only P2 with RBS and pLas with RBS had the correct size
The same experiment with the ssUSP45DspB was performed again, but again no positive results were obtained
PCR was performed with the synthetic ssUSP45DspB gene, resulting in ssUSP45 and ssUSP45DspB without the His-tag
September 1 - September 5
Obtaining ssUSP45DspB (BBa_K1365111) as a BioBrick in the pSB1C3 vector and ssUSP45Aiia (BBa_K1365169) as a BioBrick in pSB1C3 vector
Two PCR reactions directly on the products were performed
The products were ligated with pSB1C3, and transformed to E. coli DH5α
Another PCR was done with the amplificated PCR product of ssUSPdspB, one with the signal sequence of USP45, and one with the ssUSP45DspB without His-tag
These products were also ligated with pSB1C3, and transformed to E. coli
Afterwards, Gibson assembly had been done with modified Aiia and the signal sequence of USP45 making ssUSP45aiiA, to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final Gibson product
The PCR product was checked on gel, giving positive results for the ssUSP45aiiA, then it was ligated with pSB1C3, and transformed to E. coli
Several colonies were visible on the plated transformation products
Colony PCR was done on them with the regular pSB1C3 test primers, which showed no results
The colonies were grown overnight, and the plasmids were isolated in the next week
September 8 - September 12
Analysis of the constructed Biobricks
PCR was performed directly on the ssUSP45DspB product
The plamids containing the ssUSP45aiiA was isolated our of E. coli DH5α and checked on gel, which showed some positive results for the contructs
New primer were designed, to give it another try
the first BioBricks were send for sequencing
September 15 - September 19
Obtaining the anti-Staphylococcus aureus system (p2s) and the anti-Pseudomonas aeruginosa system(pLASs)
Both systems were assembled by using Gibson assembly
Downstream of the pLas a RBS is attached together with the promoter P2 and dspB with tail PCR
A Gibson tail was attached with PCR to the P2 with RBS, dspB with RBS, aiiA and the double terminator
ssUSP45DspB, and nisA was added and incubated for 90 minutes at 50 °C
The Gibson fragment was amplified with gradient PCR
The construct was digested, checked on gel, and send for sequencing
Only 1200 base pairs of the construct could be found on gel
