--------------------------------------------------------------
This year, we wanted to continue optimising the activities of the cyclase for 5AVA, 3-hydroxybutyrate dehydrogenase (CF3HBD). Moreover, we also included lactam-binding NitR as the other potential redesigning target, which takes a critical role in ligand fishing towards the end of our project.
First, we modelled and characterised the ability of several mutants in the dry lab. Mutants were selected based on our calculations of protein stability after iterative energy minimization and simulated point mutations. Structures were further refined by using the Relax and Backrub applications of the Rosetta software suite [2]. Then, the mutants' activities were compared by QM/MM using the CP2K software package; we selected 8 CF3HBD and 4 NitR mutants to continue testing in the wet lab.
The sequences of the CF3HBD mutants are registered as BBa_K4383004 to BBa_K4383011, and the sequences of the NitR mutants are registered as BBa_K4383000 to BBa_K4383003. Standard cloning techniques were used to insert the CF3HBD and NitR mutants into pET28a and pETBlue2 plasmids respectively. The recombinant vectors, pET28a_CF3HBD and pETBlue2_NitR harbouring genes of CF3HBD and NitR respectively, were transformed in E.coli DH5α and plated on agar plates with ampicillin for pETBlue2_NitR and kanamycin for pET28a_CF3HBD. The transformed clones were screened through colony PCR and the vectors were amplified through plasmid preparation.
For expression of the mutants, the vectors pET28a_CF3HBD and pETBlue2_NitR were transformed into E.coli NiCo21 cells and plated on agar plates with their respective antibiotics. The LoFT protocol was then used to prepare cell extracts, and the proteins were purified by His-tag purification.
Due to restraints during the pandemic, we could not analyse the proteins obtained from His-tag purification by SDS-PAGE for their enzymatic properties. We originally planned to analyse protein yields by chemical methods such as IR titration to measure the reaction with substrates.
We have learnt how to optimise the factors in the LoFT protocol, such as shaking time, speed, and temperature required to achieve the best yield in our laboratory [3].
We plan to finish our final step of confirming protein quality by SDS-PAGE. Our proposed implementation for NitR is to use protein ligand fishing to bind NitR to magnetic beads, and use the protein-bead complex to capture cyclized valerolactam from cell extract.
In the first year of our project, we planned using the INTEGRATE System and our shuttle vector panS-loxP-MCS to insert the genes of AK, DavB and DavA into the chromosome of S.elongatus UTEX 2973 [4]. However, in the process of cloning the pSPIN plasmid for the INTEGRATE System, we discovered that cell counts were quite low, likely due to the large 12kb plasmid size putting too much metabolic pressure on the cells. This resulted in low plasmid yields, slowing the entire cloning process, and also indicating that the INTEGRATE System was not the most suitable choice for genomic integration in our project.
Recognizing this difficulty, we changed our method for genomic insertion of S.elongatus UTEX 2973. We instead chose the GeneArt™ Synechococcus Protein Expression Vector (pSyn_6), which allows homologous recombination of exogenous DNA into the natural site 1 of S.elongatus [5]. In our new design we ligate the genes of AK, DavB and DavA from our vector davBA-AK-pUC into pSyn_6.
davBA-AK-pUC was cloned last year through Gibson Assembly. pSyn_6 and davBA-AK-pUC were transformed into E.coli DH5α and plated on agar plates with streptomycin and ampicillin respectively. The vectors were amplified by plasmid preparation.
Due to restraints during the pandemic, we could not carry out restriction enzyme digestion and ligation for inserting the genes of AK, DavB and DavA from our vector davBA-AK-pUC into pSyn_6. We also could not test electroporation of pSyn_6 into S.elongatus UTEX 2973 to see its resistance to streptomycin. However, we did succeed in transformation by electroporation protocol on S.elongatus UTEX 2973 last year.