Difference between revisions of "Part:BBa K3490031"
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Revision as of 13:16, 26 October 2020
The toxin-antitoxin system: hicA-hicB
Overview
During the experiment process, we had difficulty constructing both plasmid. With the doubt that the EL222-FLP system might be harmful to the growth of bacteria, we changed the design of blue-sensitive protein EL222 into thermo-sensitive protein CI857 on the plasmid pCP20 provided by our PI, Prof. Ng (Fig.4).
Fig.3. The plasmid map which changes the toxin-antitoxin gene into fluorescent genes
Fig.4. The heat-activated plasmid pCP20
The thermo-sensitive system: pCP20
Therefore, we employed heat-activation, as our new method to resuscitate the bacteria. The thermo-sensitive system can be activated through the degradation of CI857 protein when the temperature rises to 36-40 ºC.[5] This time, the experiments of ligation and transformation were successful, but the color of GFP were not observed in the bacteria after transformation. Suspecting that the sequence might be mutated, we sent plasmid for DNA sequencing. As we expected, the sequencing region matched poorly to our GFP-OFP design. As shown in Fig. 4, one of the FRT sequences is severely mutated next to the promoter J23100(Fig.5). This suggests that our design had violated some fundamental principle. Since the FLP recombinase works by twisting the DNA to bring two FRT sites together and nicking them to generate insertion or deletion. We speculated that the distance between the two FRT sites is insufficient for the two FRT sites to form a loop in vivo. We then check for the optimal distance between two FRT sites for the FRT-FLP system to function. Previous report states that the optimal distance for the recombination to take place is around 200bp in vitro[6], while our original design between two FRT sites is only 160bp, this may be the cause of malfunction.
Nevertheless, we had to come up with a new design.
Fig.5. The sequencing result of sfGFP-FRT-J23100-FRT-OFP, J23100 is highlighted, and the red arrow indicates FRT sequence flanking J23100
The change of FRT mechanism
FRT sites can either change the orientation of the gene or delete the gene depending on the orientation of the two FRT sequences[7]. Since the design of changing the promoter direction failed, we choose to delete the gene, which is more common in general lab settings. For the new design, we still used the plasmid pCP20 for heat-activation, but we put hicA gene between the FRT sites for deletion on the plasmid pSB1C3 (Fig 6.a), and the plasmid pSB3K3 with hicB gene was also designed to continuously produce HicB (Fig 6.b). Because pSB3K3 is a low copy number plasmid, the expression of HicB is less than HicA, therefore, the bacteria will hibernate. After heat activation, hicA gene will be deleted while hicB gene is still expressed, so the bacteria can resuscitate. For these three plasmids, we can regulate the growth of bacteria, meeting our need to design a growth switch.
Fig.6.a. The design of BBa_K3490030
Fig.6.b. The design of BBa_K3490031
The experiment of kanamycin-resistant gene deletion: Prove of design
Due to time constraint, we can only demonstrate this model using pKD3 plasmid, which carries a kanamycin-resistance gene flanked between two FRT sites provided by our PI, Prof. Ng. If the bacteria cannot survive at LB plate with kanamycin after heat-activation, we can demonstrate that the gene between the FRT sites was deleted, indicating that our design is feasible. For the experiment, we co-transformed pKD3 and pCP20 into E. coli DH5α, and cultured a colony in 37℃ for 8 hr and transfer to 42℃ for 1 hr to heat-activate the bacteria, and then spread on the LB plate (dilute 105 times) and culture for 12 hr. After the colony grew, we screened the colony on a kanamycin plate to check whether the bacteria will grow or not. Results show that most of the bacteria can not survive on the Kanamycin plate but can still survive on the LB plate without kanamycin (Fig.7, 8.), which means the antibiotic-resistance gene was deleted successfully. With this data, we are convinced that the design of pSB3K3 with hicA gene is feasible.
Fig.7. The bacteria can grow on the LB plate without antibiotics (the left one), but when we pick the colony from it to the kanamycin plate (the right one), there is almost no colony on the plate.
Lane1: the uncut plasmid with activation of the FRT system
Lane2: the plasmid cut by HindIII with activation of the FRT system
Lane3: the uncut plasmid without activation of the FRT system
Lane4: the plasmid cut by HindII without activation of the FRT system
LaneM: the marker
Fig.8. The plasmid pattern before and after activating FRT genes checked by DNA gel electrophoresis.
Reference
[1]Guglielmini J, Van Melderen L. Bacterial toxin-antitoxin systems. Mobile Genetic Elements. 2011;1(4):283-306.
[2]Jørgensen MG, Pandey DP, Jaskolska M, Gerdes K. HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea. Journal of Bacteriology. 2008;191(4):1191-1199.
[3]Motta-Mena LB, Reade A, Mallory MJ, et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology. 2014;10(3):196-202.
[4]Branda CS, Dymecki SM. Talking about a Revolution. Developmental Cell. 2004;6(1):7-28.
[5]Jechlinger W, Szostak MP, Witte A, Lubitz W. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene expression in Escherichia coli. FEMS Microbiology Letters. 1999;173(2):347-352.
[6]Ringrose L. Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances. The EMBO Journal. 1999;18(23):6630-6641.
[7]Park Y-N, Masison D, Eisenberg E, Greene LE. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae. Yeast. 2011;28(9):673-681.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 109
Illegal BsaI.rc site found at 149