Team:Shanghai HS ID/Results
Results
1. DNA Profiles
Figure 1. DNA profile of the plasmid PLCNICK
Figure 2. DNA Profile of plasmid PTB165 which was electrotransformed into L. casei for transformation
efficiency test
Figure 3. DNA Profile of LSEI-2094+ upstream and downstream homologous arms
2. Electrophoresis result after PCR
Figure 4. Electrophoretogram of pLCNICK PCR resiult
1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme
digestion, 6~7
are upstream
homologous arms after PCR, 8~9 are downstream homologous arms after PCR.
3. Verification result of colony PCR
Figure 5. PCR verification result
As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are
consistent with the DNA
profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the
plasmid in E. coli.
4. Transformation test result
Graph 1. Comparison between the wild L. caseil and modified L. casei in transformation
After we electrotransformed the plasmid pIB165 as the foreign DNA to test the
transformation efficiency of our modified L. casei, it took several days to culture and finally saw the
comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation
efficiency with remarkably more colonies than the wild (Wild).
Figure 6. Histogram comparison between wild strain and KO strain
In addition, we measured OD600 of these strain groups which were pre-spreaded plates with
different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig.
6).
In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas.
In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas.
5. Future plan and our concern
As we have primarily tested the transformation effiency of our modified L. casei, we would think about to
further explore the expression performance of the foreign plasmid as well. Besides, we have built the
partnership with EV71 Terminator (2021 iGEM team Shanghai_Metropolis ) who we will work together to develop
a HFMD Oral Vaccines using L. casei as the live carrier. Therefore, in next step we will electrotransform
the plasmid constructed by EV71 Terminator into our modified L. casei and test the expression performance of
this recombinant L. casei.
About our modified L. casei as well as its derived recombinant bacterium, we also take the safety and stability into acount, it would be important for us to do more literature research and seek more professional advice on future development of our product before we step into the real application.
About our modified L. casei as well as its derived recombinant bacterium, we also take the safety and stability into acount, it would be important for us to do more literature research and seek more professional advice on future development of our product before we step into the real application.