Difference between revisions of "Team:iTesla-SoundBio/design"
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<div id="constructdesign"> | <div id="constructdesign"> | ||
<p class="text"> | <p class="text"> | ||
| − | Construct design: <br> | + | <u>Construct design: </u><br> |
The goal for our project is to insert Factor C gene into the chromosomal DNA of B. Subtilis, and have it express this Factor C protein. At the same time, to fulfill iGEM competition requirements, we also need to insert Factor C gene into E.coli plasmid pSB1C3 and submit it as a biobrick for iGEM competition. | The goal for our project is to insert Factor C gene into the chromosomal DNA of B. Subtilis, and have it express this Factor C protein. At the same time, to fulfill iGEM competition requirements, we also need to insert Factor C gene into E.coli plasmid pSB1C3 and submit it as a biobrick for iGEM competition. | ||
<br> | <br> | ||
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<center><img src="https://static.igem.org/mediawiki/2018/c/c6/T--itesla-soundbio--twofragments.png", width=80%></center> | <center><img src="https://static.igem.org/mediawiki/2018/c/c6/T--itesla-soundbio--twofragments.png", width=80%></center> | ||
<p class="text"> | <p class="text"> | ||
| − | Biobrick: <br> | + | <u>Biobrick: </u><br> |
Factor C in E.coli pSB1C3 | Factor C in E.coli pSB1C3 | ||
We couldn't order Factor C from factory because it is too large (about 3000 base pairs https://benchling.com/itesla-soundbio/f/gPUeHZXf-drafts/seq-Jq87odxl-psb1c3factor-c/edit), so we order it as two fragments, FC1 and FC2. | We couldn't order Factor C from factory because it is too large (about 3000 base pairs https://benchling.com/itesla-soundbio/f/gPUeHZXf-drafts/seq-Jq87odxl-psb1c3factor-c/edit), so we order it as two fragments, FC1 and FC2. | ||
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<div id="Integration"> | <div id="Integration"> | ||
<p class="text"> | <p class="text"> | ||
| − | Integration: <br> | + | <u>Integration: </u><br> |
Factor C in B. Subtilis Chromosomal DNA. | Factor C in B. Subtilis Chromosomal DNA. | ||
We then digest full Factor C from Biobrick, and insert it into pAX01, a B. Subtilis integration factor. Then, we transform B. Subtilis with pAX01. After plasmid pAX01 enters B. Subtilis, there is a chance that it will integrate into chromosomal DNA. | We then digest full Factor C from Biobrick, and insert it into pAX01, a B. Subtilis integration factor. Then, we transform B. Subtilis with pAX01. After plasmid pAX01 enters B. Subtilis, there is a chance that it will integrate into chromosomal DNA. | ||
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<div id="expression"> | <div id="expression"> | ||
<p class="text"> | <p class="text"> | ||
| − | Expression | + | <u>Expression</u> |
Factor C protein extract: <br> | Factor C protein extract: <br> | ||
B. Subtilis are used to express Factor C because they are gram positive and will not damage the Factor C protein. In contrast, E. coli is gram negative and will cleave FC protein. After B. Subtilis express and produce Factor C, we can then extract Factor C protein for further experiment. | B. Subtilis are used to express Factor C because they are gram positive and will not damage the Factor C protein. In contrast, E. coli is gram negative and will cleave FC protein. After B. Subtilis express and produce Factor C, we can then extract Factor C protein for further experiment. | ||
Revision as of 01:29, 1 October 2018
Engineer the Future
That Prosperity and Environmentalism Coexist
Construct Design
Biobrick:
Factor C gene in E. coli pSB1C3
Integration:
Factor C in B.Subtilis Chromosomal DNA
Expression:
Factor C protein verification
Construct design:
The goal for our project is to insert Factor C gene into the chromosomal DNA of B. Subtilis, and have it express this Factor C protein. At the same time, to fulfill iGEM competition requirements, we also need to insert Factor C gene into E.coli plasmid pSB1C3 and submit it as a biobrick for iGEM competition.
We decided to insert Factor C gene into E.coli plasmid pSB1C3 first; then, integrate Factor C gene into the chromosomal DNA of B. Subtilis using pAX01 integration factor (a B. Subtilis specific plasmid).
Biobrick:
Factor C in E.coli pSB1C3
We couldn't order Factor C from factory because it is too large (about 3000 base pairs https://benchling.com/itesla-soundbio/f/gPUeHZXf-drafts/seq-Jq87odxl-psb1c3factor-c/edit), so we order it as two fragments, FC1 and FC2.
First, insert FC1 into pSB1C3. Then, insert FC2 into the plasmid, too. This pSB1C3 with full Factor C will be submitted for biobrick.
Integration:
Factor C in B. Subtilis Chromosomal DNA.
We then digest full Factor C from Biobrick, and insert it into pAX01, a B. Subtilis integration factor. Then, we transform B. Subtilis with pAX01. After plasmid pAX01 enters B. Subtilis, there is a chance that it will integrate into chromosomal DNA.
Expression
Factor C protein extract:
B. Subtilis are used to express Factor C because they are gram positive and will not damage the Factor C protein. In contrast, E. coli is gram negative and will cleave FC protein. After B. Subtilis express and produce Factor C, we can then extract Factor C protein for further experiment.