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Results

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Part Plasmids Assembly Plasmids Electroporations Conjugations

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Table 1: This is the number of parts organized by type that have been made thus far. All contribute to the BHR kit and many are included in full assemblies.

There are millions of species of bacteria¹, 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 researchers². Synthetic biologists take great interest in engineering such bacteria, in an attempt to harness their natural abilities³. 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 (GGA) to build plasmids. GGA 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 standardized⁴. 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.

Figure 1: This figure shows how the kit is used. A mixture of part plasmids is transformed into a sample of host bacteria, then it is plated on selective media. The colonies will express their colored reporter gene, indicating which plasmids are maintained by the host. If there are colonies, but no expression is apparent, then the colonies can be sequenced to determine which ORI allowed replication. Knowing which ORIs a bacteria can use to maintain plasmids are important for researchers building their own plasmid for non-model bacteria.

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 GGA 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 GGA's modular design that maintains directionality.

References

1. Amann, R. and Rosselló-Móra, R. (2016) After All, Only Millions? American Society for Microbiology, 7 (4): 1-2.
2. Nemergut, et al. (2011) Global patterns in the biogeography of bacterial taxa. Environmental Microbiology, 13: 135-144.
3. Liu, H. and Deutschbauer, A. (2018) Rapidly moving new bacteria to model-organism status. Current Opinion In Biotechnolodgy, 51:116–122.
4. Jain, A.,& Srivastava, P. (2013) Broad host range plasmids. FEMS Microbiol Lett, 348: 87–96. 10