To avoid any possible contamination by the biosensor itself, we are designing a kill switch system, consisting of innate CcdA/CcdB type II toxin-antitoxin system to regulate the number of biosensors [1] in the whole expression cassette. ccdB toxin is constantly expressed, if QscR cannot detect any AHL molecules, the antitoxin CcdA will no longer be released.
We adapted the kill-switch system to prevent environmental contamination. We used SnapGene to build the CcdA and CcdB genes. The two sequences (BBa_K4823010 and BBa_K4823011) are uploaded to the registry. Due to limited time we do not have time to insert the genes by standard cloning techniques.
We have designed one pUC_idt_PA1897_EGFP_Kan Golden Gate mutant plasmid. We aim to design a plasmid with no leaky expression of EGFP. To do this, we have to delete lac operon from the plasmid. The lac operon is the innate operon of PA1897. It ensures normal expression of protein EGFP whenever the E. coli cell is living. This is undesirable because it intervenes in our wanted regulated quantity of EGFP for the purpose of precision.
The sequence of the pUC_idt_PA1897_EGFP_Kan Golden Gate mutant is registered as BBa_K4823012. We have knocked out lac operon from pUC-IDT-PA1897-EGFP and thus created a mutant plasmid, by using New England Biolabs Q5® Site-Directed Mutagenesis Kit. The PA1897 mutant is co-transformed, along with pET_21a (+) _qSCR in E.coli BL21 and plated on agar plates with ampicillin for pET_21a(+)_qSCR and kanamycin for pUC_idt_PA1897_EGFP_Kan Golden Gate mutant. The plate shows positive results for bacteria culture, indicating the success of co-transformation.
Although we failed to confirm the two plasmids’ band size by restriction digestion, we did manage to confirm by colony PCR. The sequencing result also confirms that pUC_idt_PA1897_EGFP_Kan Golden Gate mutant has the correct sequence.
We ran a blast and observed only 50 percent of the pET_21a (+) _qSCR alignment with our gene of interest. We did troubleshooting for every step. We learned about the importance of annealing temperature as well as extension time in PCR. Since we have used the Nde1 restriction site, we observed its a poor cutter so we extended the time of incubation at each step. We were recommended for overnight ligation.
In our project, we will use two plasmids, pET21a(+)_idt_qSCR and PA1897_EGFP mutant. We will design our system in such a way that the two separate plasmids will be co - transformed into the E. coli BL21 cell. They would function separately, while connected by the expression of qSCR protein for upregulation. The sequences of the plasmid are designed in Snapgene. When transforming the two plasmids, we have used double selection markers, ampicillin for pET_21a(+)_qSCR and kanamycin for pUC_idt_PA1897_EGFP_Kan Golden Gate mutant.
The recombinant plasmids pUC_EGFP and pET_qSCR are co-transformed using standard transformation protocol into E.coli BL21 cells. The transformation of two plasmids was done within the same step. On the agar plates, positive bacteria cultures are shown.
The sequencing result of pUC_EGFP is and the colony PCR of pET_qSCR plasmids are shown positive. The co-transformation succeeded.
We had to standardize the protocols while transforming two plasmids in BL21 cells as unlike before we are using two different antibiotics. The choice of strain changes from DH5ɑ to BL21, as the latter has a higher capacity of protein expression. At a later stage if we are to carry out GFP quantification tests, the BL21 would be more suitable.
Fraikin, N., Goormaghtigh, F., & Van Melderen, L. (2020). Type II Toxin-Antitoxin Systems: Evolution and Revolutions. Journal of bacteriology, 202(7), e00763-19. https://doi.org/10.1128/JB.00763-19