Team:HK SSC/Results
Results
Work we have done:
- Cloned three plasmids for plasmid preparation: pSPIN, panS-loxP-MCS, davBA-AK-pUC
- E.coli DH5α transformed with pSPIN showed resistance to streptomycin
- Cloned panS-loxP-MCS by Gibson Assembly
- Cloned davBA-AK-pUC by Gibson Assembly
- E.coli DH5α transformed with panS-loxP-MCS showed resistance to kanamycin
- E.coli DH5α transformed with davBA-AK-pUC showed resistance to ampacillin
- Modeling of cyclase CF3BD
- Cultivated S.elongatus UTEX 2973 cells
- transformed S.elongatus UTEX 2973 cells with panS-loxP-MCS
- showed resistance to kanamycin after antibiotic challenge
Discussion
We were able to obtain enough plasmid yield for transformation of E.coli DH5α cells, confirming the presence of the plasmids. However, due to a limitation of laboratory equipment, our plasmid preparation yields were constantly low. This affects the transformation into UTEX 2973 as over 4 µg of DNA (size ~10kb) was reported to be required for a successful transform. We also faced difficulty during transformation, needing to repeat many weeks before obtaining a definite result. pSPIN’s large plasmid size may have also contributed to its low plasmid and transformation yield.
In the coming year, we will be receiving new lab equipment for performing precise procedures. We will be testing the efficiency and function of the plasmids by cloning them with reporter genes. The efficiency and function of AK, DavB, DavA and CF3BD will also be assayed by expression analysis and evaluating yield of lysine, 5-AVA and valerolactam.