"
Team:USyd-Australia/Project/Design
From 2014.igem.org
| Line 9: | Line 9: | ||
like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2> | like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for <a href="https://2014.igem.org/Team:USyd-Australia/Project/Protocols#gibson">Gibson Assembly</a>.</p><br><h2> </h2> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width=" | + | <h3>Step2: Express Integrase</h3> |
| - | <br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p><br><h2> </h2> | + | <img src="https://static.igem.org/mediawiki/2014/1/1f/USyd-Australia_Integron_Design-Express_Integrase.png" width="340px" align="left"> |
| + | <br> <p>As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part <a href="https://2014.igem.org/Team:USyd-Australia/pUS203">pUS203</a> is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from <a href="http://parts.igem.org/Part:BBa_K731201">K731201</a> which allows us to turn on transcription of the Integrase under the activation of arabinose.</p> | ||
| + | <p> By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop excision of cassettes from the array from occurring. </p> | ||
| + | <br><h2> </h2> | ||
<img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2014/5/55/USyd-Australia_Integron_design-add_cassettes.png" width="60%"> | ||
Revision as of 23:01, 17 October 2014
 |
Project Design
Step 1: Produce Cassettes
Gene Cassettes are the carriers of genetic information in our design. They are small, modular chunks of DNA containing a gene flanked by AttC sites. Cassettes, unlike plasmids, are non-replicative and so do not require extra genes to modulate or initiate replication. To create a cassette simply requires the use of primers to create half AttC sites at either end and circularizing the product by using Enzymatic Ligation Assisted by Nucleases (ELAN). This is a reaction in which ligation and enzymatic digestion of compatible but non-identical ends, like XbaI and SpeI, occurs simultaneously. This should result only in stable single-cassette circles or concatemers of several cassettes. For systems that already contain an intact AttC site, XbaI and SpeI sites can be introduced by PCR for ELAN, or overlapping ends can be introduced by PCR for Gibson Assembly.
Step2: Express Integrase
As gene cassettes are non-replicative it is important that they are incorporated into an AttI site before the cell divides and the cassettes face the possibility of ejection. Therefore it is vital to have a controllable Integrase gene which can be turned on prior to the addition of gene cassettes to ensure that the cassettes are integrated before they are lost. Part pUS203 is our submission BioBrick which encodes the controllable Integrase system. We are using the araC-pBAD system from K731201 which allows us to turn on transcription of the Integrase under the activation of arabinose.
By controlling integrase expression with arabinose, we are also able to turn off integrase, which would stop excision of cassettes from the array from occurring.
As gene cassettes enter the cell site the integrase acts to cause specific recombination between the AttC site on the cassette and an AttI site which may be present on a plasmid or the bacterial chromosome. The cassette forms a holiday junction at the recombination site.
Now that the required cassette is present in the array we can induce expression of the genes from an upstream promoter to the cassette array. A single promoter can read through multiple cassettes if a terminator is not present, allowing multiple genes to be expressed from a single signal
With thanks to:
