Difference between revisions of "Team:Austin UTexas/Description"

Problem: Non-Model Organisms are Hard to Work With!

However, different genetic parts, such as promoters or antibiotic resistance genes can cause a plasmid that can be maintained in model organisms, to not be maintained in non-model ones. Broad host range plasmids are useful for this because they contain the optimal combination of parts that can replicate and be maintained in many different bacteria.

Solution: Use a Broad Host Range Library!

The Broad Host Range project aims to create a series of part plasmids whose parts can be relatively quickly assembled using golden gate assembly to one plasmid, called an assembled cassette plasmid. A cassette plasmid is composed of many different parts:

Cassette plasmids are assembled using a technique called golden gate assembly. Golden gate assembly utilizes the advantage of type IIs restriction enzymes. Type IIs restriction enzymes cleave next to the stretch of DNA they recognize so that the same recognition site is not preserved. Our library’s cassette plasmids have been designed to be recognized once by the restriction enzyme, Bsa1. To transform the desired host with your own DNA construct, perform a golden gate assembly with restriction enzyme BsmB1 to insert the DNA construct into many different versions of cassette plasmids. Then, transform a sample of the host organisms with the all the altered cassette plasmids. Because you are using BHR plasmids, it will be more likely that your transformants can proliferate.

Part Number	Part Type
Type 1	Assembly Connector
Type 2	Promoter/RBS
Type 3	Coding Sequence
Type 4	Terminator
Type 5	Assembly Connector
Type 6	Barcode
Type 7	Origin of Transfer
Type 8a	Origin of Replication
Type 8b	Antibiotic Resistance

Part types 1, 2, 4, 5, 6, 7 and 8 are important for plasmid replication. Part type 3 is the plasmid part/gene to be incorporated in the non-model organism. Part type 8b helps maintain a plasmid. When a non-host organism needs to be transformed, many of cassette plasmids of different genetic parts can be transformed into the microorganism. The plated surviving organisms’ DNA can be sequenced to reveal which assembled cassette plasmids can be maintained and replicated.