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
This research aimed to (1) design a small library of level 0 parts for filamentous fungi, which consisted in two promoters, one fluorescent reporter, one terminator, an antibiotic resistance cassette and the Autonomously maintained in Aspergillus sequence, all of them in the Phytobrick standard from iGEM, (3) improve the iLOV fluorescent protein by adding the phytobrick standard (3) assemble expression cassettes with the mentioned parts, (5) transform P. roqueforti and (5) characterise the expression of the designed constructs .
- Construction and submission of LV0 parts for filamentous fungi: PgdaA constitutive promoter (BBa_K1968009), PglaA inducible promoter (BBa_K1968013), Tcyc terminator (BBa_K1968014), Phleomycin antibiotic resistance cassette (BBa_K1968015) Improved iLOV in phytobrick standard (BBa_K1968016)
- Design combinatorial gblocks for the AMA1 sequence to be assembled by Gibson
- Demonstration of low-efficiency PEG-mediated transformation in P. roqueforti
- A novel demonstration of low-efficiency electroporation-mediated transformation in P. roqueforti
- Unable to experimentally characterise the PglaA (BBa_K1968009) and PglaA (BBa_K1968013) promoters
- Unsuccessful gibson assembly for the AMA1 sequence
- Unable to define a reliable and efficient protocol for transformation of P. roqueforti
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
