Difference between revisions of "Team:Duke/Design"
| Line 11: | Line 11: | ||
<p>In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the BAPT gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-HC-Amp Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid </p> <br> <br> | <p>In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the BAPT gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-HC-Amp Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid </p> <br> <br> | ||
| − | <h4><center>pSMART-HC-Amp-BAPT</h4></center><br> | + | <h4><center>pSMART-HC-Amp-BAPT Plasmid Map</h4></center><br> |
| − | <center><img src="https://static.igem.org/mediawiki/parts/ | + | <center><img src="https://static.igem.org/mediawiki/parts/0/09/T--Duke--pSMART-HC-Amp-BAPT_Plasmid_Map.jpg" style="width:400px;height:280px;"></center> |
<br><br> | <br><br> | ||
<p>This year we amplified the BAPT gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested BAPT and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-BAPT. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing. | <p>This year we amplified the BAPT gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested BAPT and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-BAPT. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing. | ||
| − | <h4><center>PSB1C3-BAPT</h4></center><br> | + | <h4><center>PSB1C3-BAPT Plasmid Map</h4></center><br> |
<center><img src="https://static.igem.org/mediawiki/parts/f/f9/T--Duke--PSB1C3-BAPT_Plasmid_Map.jpg" style="width:400px;height:280px;"></center> | <center><img src="https://static.igem.org/mediawiki/parts/f/f9/T--Duke--PSB1C3-BAPT_Plasmid_Map.jpg" style="width:400px;height:280px;"></center> | ||
<br><br><br><br> | <br><br><br><br> | ||
| Line 24: | Line 24: | ||
<p>In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the TAX10 gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-LC-Kan Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid </p> <br> <br> | <p>In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the TAX10 gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-LC-Kan Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid </p> <br> <br> | ||
| − | <h4><center>pSMART-LC-Kan-TAX10 Plasmid</h4></center><br> | + | <h4><center>pSMART-LC-Kan-TAX10 Plasmid Map</h4></center><br> |
| − | <center><img src="https://static.igem.org/mediawiki/parts/ | + | <center><img src="https://static.igem.org/mediawiki/parts/2/26/T--Duke--pSMART-LC-Kan-TAX10_Plasmid_Map.jpg" style="width:400px;height:280px;"></center> |
<br><br> | <br><br> | ||
<p>This year we amplified the TAX10 gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested Tax10 and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-TAX10. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing. | <p>This year we amplified the TAX10 gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested Tax10 and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-TAX10. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing. | ||
| − | <h4><center>PSB1C3-TAX10</h4></center><br> | + | <h4><center>PSB1C3-TAX10 Plasmid Map</h4></center><br> |
| − | <center><img src="https://static.igem.org/mediawiki/parts/f/f9/T--Duke--PSB1C3- | + | <center><img src="https://static.igem.org/mediawiki/parts/f/f9/T--Duke--PSB1C3-Tax10_Plasmid_Map.jpg" style="width:400px;height:280px;"></center> |
<br><br> | <br><br> | ||
Revision as of 20:50, 17 October 2018
Design
BAPT
In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the BAPT gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-HC-Amp Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid
pSMART-HC-Amp-BAPT Plasmid Map
This year we amplified the BAPT gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested BAPT and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-BAPT. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing.
PSB1C3-BAPT Plasmid Map
BAPT
In 2016, the Duke iGEM team ordered a gBlock from IDT containing the dna code for the TAX10 gene as well as 20 base pair overhangs with the Lucigen vector, pSMART-LC-Kan Plasmid. Linearized pSMART and gBlock underwent Gibson Assembly to create a circular plasmid
pSMART-LC-Kan-TAX10 Plasmid Map
This year we amplified the TAX10 gene from the above plasmid using primers SL1 and SR2. The PCR was then digested with DPN1 to remove the pSMART vector and digested with XbaI and SpeI. Likewise, PSB1C3-RFP was digested with XbaI, SpeI, and CIP to prevent autoligation. The digested Tax10 and PSB1C3 were then ligated with a 6:1 molar ratio to obtain PSB1C3-TAX10. White colonies were picked from the transformation and confirmed with colony PCR and later sequencing.
PSB1C3-TAX10 Plasmid Map
Modular Design and Path Optimization
Maria The end goal of the project was to create a linear stretch of DNA containing all five genes in the pathway that could be recombineered into the E. coli genome. In creating this pathway, each gene was viewed as a separate "building block" that would be linked with the other blocks to create a complete pathway. Within one gene's "building block," there were several components: the 5' linker arm, the T7 promoter, the coding sequence of the gene, and the 3' linker arm.
Promoter Optimization Experimental Design
Maria The design of the following elements of the project was laid out, but not experimentally implemented. LINE BREAK HOW DO I DO THOSE. Each of the 5 genes in the pathway was designed to be fitted with a different T7 bacteriophage promoter.
Design
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
This page is different to the "Applied Design Award" page. Please see the Applied Design page for more information on how to compete for that award.
What should this page contain?
- Explanation of the engineering principles your team used in your design
- Discussion of the design iterations your team went through
- Experimental plan to test your designs