Composite

Part:BBa_K3490030

Designed by: Ren Hao Tsai   Group: iGEM20_NCKU_Tainan   (2020-10-23)


The toxin-antitoxin system : hicA-hicB


The final version of growth switch

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.1. The plasmid map which changes the toxin-antitoxin gene into fluorescent genes


Fig.2. 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.[1] 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[2], 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.3. 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[3]. 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.4. The design of BBa_K3490030


Fig.5. 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.6. 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.7. The plasmid pattern before and after activating FRT genes checked by DNA gel electrophoresis.



Result: Predicting success


Reference

[1]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.
[2]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.
[3]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:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1115
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1055
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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