Difference between revisions of "Team:Austin UTexas/Description"
| Line 53: | Line 53: | ||
<h3>Solution: Use a Broad Host Range Library!</h3> | <h3>Solution: Use a Broad Host Range Library!</h3> | ||
| − | <p style=font-family:' | + | <p style=font-family:'Ailerons'>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:</p> |
<p>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 recognition site is preserved, but it can create unique overhangs. These unique overhangs are what gives Golden Gate Assembly its ability to maintain directionality when assembling multiple parts. The first step is to use the restriction enzyme BsmBI to create part-plasmids from parts of interest and entry vectors. Then assembly plasmids can be constructed from various part-plasmids using the restriction enzyme BsaI in a one-pot reaction. Lastly, these plasmids can be transformed into the organism of interest to test its functionality. Because you are using BHR plasmids, it will be more likely that your transformants can proliferate.</p> | <p>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 recognition site is preserved, but it can create unique overhangs. These unique overhangs are what gives Golden Gate Assembly its ability to maintain directionality when assembling multiple parts. The first step is to use the restriction enzyme BsmBI to create part-plasmids from parts of interest and entry vectors. Then assembly plasmids can be constructed from various part-plasmids using the restriction enzyme BsaI in a one-pot reaction. Lastly, these plasmids can be transformed into the organism of interest to test its functionality. Because you are using BHR plasmids, it will be more likely that your transformants can proliferate.</p> | ||
Revision as of 17:51, 29 June 2018
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 recognition site is preserved, but it can create unique overhangs. These unique overhangs are what gives Golden Gate Assembly its ability to maintain directionality when assembling multiple parts. The first step is to use the restriction enzyme BsmBI to create part-plasmids from parts of interest and entry vectors. Then assembly plasmids can be constructed from various part-plasmids using the restriction enzyme BsaI in a one-pot reaction. Lastly, these plasmids can be transformed into the organism of interest to test its functionality. 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, several assembly plasmids of different genetic parts can be transformed into the microorganism. The plated surviving organisms’ DNA can be sequenced to reveal which assembly plasmids can be maintained and replicated.