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

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Construct design:  
 
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.  
 
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).  
 
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
 
Biobrick
 
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.  
 
First, insert FC1 into pSB1C3. Then, insert FC2 into the plasmid, too. This pSB1C3 with full Factor C will be submitted for biobrick.
 
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  
 
Integration  
 
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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Expression
 
Expression
 
Factor C protein extract
 
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.     
 
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 00:39, 30 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

Graphic Summary
Construct Design
Biobrick:
Factor C gene in E. coli pSB1C3
Integration:
Factor C in B.Subtilis Chromosomal DNA
Expression:
Factor C protein verification
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.