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

Line 55: Line 55:
 
<p style=font-family:'Montserrat'>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 style=font-family:'Montserrat'>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 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.</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>
  
 
<div class="row">
 
<div class="row">
Line 116: Line 116:
  
  
−
<p>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.</p>
+
<p>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.</p>
  
  

Revision as of 16:02, 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.

Figure taken from A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly 2015 Lee, Et. al
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.