Team:Austin UTexas/Results
Results
There are millions of species of bacteria, few of which are well characterized. However, these non-model organisms perform cellular processes and are native to harsh biomes, and this makes them of great interest to researchers. Synthetic biologists take great interest in engineering such bacteria, in an attempt to harness their natural abilities. Because finding the best way to genetically modify an organism can be a laborious and resource-demanding process, we have developed a kit of modular plasmids, designed to quickly test multiple broad host range origins of replication (ORIs). To do this, we used a cloning technique called Golden Gate Assembly to build plasmids. Golden Gate Assembly uses Type IIs restriction enzymes, which allows researchers to pick the overhang each cut produces. Therefore, many parts can be assembled together at once, in a specific order. The plasmids are minimal and standardized. They include barcode sequences and reporter genes to indicate which plasmid is which.
We developed a reaction where a mixture of various plasmids with different origin of replications is transformed into a single sample of host bacteria. The colored reporters allow the researcher to quickly determine which origin of replication allow the host to replicate the plasmid. However, if the reporters are not expressed, there are included primers that sequence the barcode region of the plasmid, allowing for complete verification of which plasmid(s) worked.
After determining which Origin of Replication works best in their organism of interest, the researchers can then utilize our repository of dried GGA part plasmids to create their own assembly plasmids. Due to the standardization of our kit and Golden Gate Assembly as a whole, users of our kit can create their own part plasmids and use them with the provided parts. For example, if a team wanted to produce a certain gene X in their host organism, they could use PCR to add on the proper overhangs to the gene to create a part plasmid, and then create their own assembly plasmid. All of this is possible due to Golden Gate Assembly's modular design that maintains directionality.
In the kit, we include:
- Part Type 1 Connector: x
- Part Type 2 Promoter: 7
- Part Type 3 Coding Sequence: 11
- Part Type 4 Terminator: x
- Part Type 5 Connector: x
- Part Type 6 Barcode: 7
- Part Type 7 Origin of Transfer: 1
- Part Type 8a Origin of Replication: 4
- Part Type 8b Antibiotic Resistance: 7
- Part Type 1-5 Connector: 1
- Part Plasmid Entry Vector: 3
- Assembly Plasmids: 4
- Barcode Primers: 2