Cycle 1:
Design
Our antibodies are composed of inserts including a ribosomal binding site (RBS), the antibody coding sequence, and a terminator. Non-illegal restriction sites were also placed in the middle of each component mentioned above. Initially we have decided to use a low-copy plasmid of pSB3K3 for our vector due to its ability to lower bacterial burden so as to uplift the growth rate of the strain. This is a crucial factor for us to obtain pure antibodies and hamper over-expression of antibodies instead of antibiotic resistance genes.
Build
We carried out restricted digestion to generate compatible end to ligase phosphodiester bond our insert into the plasmid pSB3K3 and upon transformation to transfer the recombinant plasmid to the vector bacteria and grow bacteria on agar plate with kanamycin.
Test
Nevertheless, experimental results unveiled that the expression level was extremely low. The transformation efficiency of pSB3K3 was also poor as shown from the data. There was miniscule growth of bacteria with the plasmid inserted as no colonels were seen on the plate.
Learn
We discerned that as pSB3K3 has kanamycin resistance, the selection of bacteria containing pSB3K3 has to use kanamycin but at the same time it will inhibit protein expression level. We then further delved into the issue by in-depth research and we came across the 2022 Virginia iGEM team that confronted the same challenge and obstacle as us. Based on the experimental results from the team and other scientific journals, they proved our previous direction to be mistaken as we should opt for vectors with a higher expression level instead of pSB3K3.
Cycle 2:
Design
We have now opted for the pET-21b expression vector which is a high-copy number plasmid and the reason for such selection is its high expression level. The selection of bacteria containing pET-21b requires the antibiotic ampicillin, it has ampicillin resistance which will not be killed by this antibiotic, and most importantly ampicillin will not inhibit protein expression level like kanamycin.
Learn
We succeeded in binding the antibody with antigen after using pET-21b. Click the results page to check out our results. For future improvement, the concentration of our antibody checked according to OD is slightly low so we should use another plasmid or modify pET-21b to higher the concentration of antibodies obtained.