Difference between revisions of "Team:iTesla-SoundBio/description"

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<center><img class="homeimg" src="https://static.igem.org/mediawiki/2018/9/9b/T--iTesla-Soundbio--Project_Description1.png" alt="DID YOU KNOW? Every year, hundreds of thousands of horseshoe crabs are caught each year and drained of up to to 30% of their blood. Why? To collect LAL, a chemical that is crucial in the detection of endotoxins in everything from drugs to medical equipment", width=60%,height:60%></center>
 
<center><img class="homeimg" src="https://static.igem.org/mediawiki/2018/9/9b/T--iTesla-Soundbio--Project_Description1.png" alt="DID YOU KNOW? Every year, hundreds of thousands of horseshoe crabs are caught each year and drained of up to to 30% of their blood. Why? To collect LAL, a chemical that is crucial in the detection of endotoxins in everything from drugs to medical equipment", width=60%,height:60%></center>
 
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Construct design:
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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.
  
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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).
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Biobrick
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Factor C in E.coli pSB1C3
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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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First, insert FC1 into pSB1C3. Then, insert FC2 into the plasmid, too. This pSB1C3 with full Factor C will be submitted for biobrick.
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Integration
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Factor C in B. Subtilis Chromosomal DNA.
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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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Expression
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Factor C protein extract
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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.   
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Revision as of 23:54, 29 September 2018

Safety Protocols
InterLab
Team Members Attributions
Human Practices Collaborations
Project Description Design Results Biobrick Parts Lab Notebook
iT-SB 2018 iT-SB 2017

Build the change

You want to see in the world

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DID YOU KNOW? Every year, hundreds of thousands of horseshoe crabs are caught each year and drained of up to to 30% of their blood. Why? To collect LAL, a chemical that is crucial in the detection of endotoxins in everything from drugs to medical equipment

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.