The aims of this project were structured as follows. (1) Create a library of parts to be characterized and submitted to the iGEM competition. (2) In an effort to have a reporter protein working in all four organisms, generate and characterize two coding sequence that, regardless of codon bias work in all four of them. (3) Confirm the correct assembly and sequence of said parts. (4) Assemble the parts into expression cassettes for characterization. (5) Transform and characterize the parts using E. coli TOP10 cells. (6) Clone constructs into the Synechocystis sp. PCC 6803 shuttle vector (pPMQAK1) and finally (7) characterize the parts using Synechocystis sp. PCC 6803.
- Designed and submited 8 phytobricks in total. Sca6-2 promoter (BBa_K1968021), L03 promoter (BBa_K1968020), reporter welov1 (BBa_K1968022), reporter welov2 (BBa_K1968023), repressor LacI (BBa_K1968024), RBS* (BBa_K1968002), promoter PA1lac0-1 (Ba_K1968000), promoter Pcpc560 (BBa_K1968001).
- Characterized Promoter Sca6-2 (BBa_K1968021)
- Predicted 2 de-optimized reporter sequences (BBa_K1968022, BBa_K1968023) that may work if used in Rhodoccocus justii, Synechocystis sp PCC 6803, Penicilium roqueforti
- Generation of transcriptional units through assembly of 4 MoClo compatible DNA parts
- Unable to experimentally characterize LacI, L03, welov1 and welov2, RBS*, promoter PA1lac0-1 (Ba_K1968000), promoter Pcpc560 (BBa_K1968001). in E. coli or Synechocystis.
- Unable to characterize parts in Synechocystis.
Rhodococcus jostii has extensive catabolic pathway to degrade variety of chlorinated compounds, such as PCBs, which makes them potential chassis for synthetic biology applications in bioremediation of toxic compounds. Nevertheless, most of the current genetic tools available for Rhodococcus still utilise traditional vector with conventional MCS. While this method has been widely used, the process is often tedious, especially when it is needed to build large metabolic pathway and characterize complex genetic elements. Therefore, we developed a set of genetic tools by adopting the Golden Gate MoClo standard to enable faster, easier synthetic biology application in R. jostii.
- Developed a set of level-1 MoClo destination vector based on the Corynebacterium - Eschericia coli shuttle vector pSRK21 by removing illegal sites and inserting standardized components (type IIs recognition sites)
- Showed a proof of concept by using the designed vectors for combinatorial assembly of compatible parts
- Showed that the designed vectors can be used for transformation in E. coli and R. jostii
- Developed a set of MoClo compatible phytobricks for R. jostii:
- Failed to change the resistance of level-1 destination vectors in order to create level-2 vectors
- Doesn’t manage to express construct in Rhodococcus
We are the University of Edinburgh Overgraduate iGEM Team, competing in the new application track in iGEM 2016. read more
School of Biological Sciences The University of Edinburgh King's Buildings Edinburgh EH9 3JF, United Kingdom
Email: edigemmsc@ed.ac.uk
