Difference between revisions of "Team:Lambert GA/Project"
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<img src="https://static.igem.org/mediawiki/2015/e/e2/Lambert_project_fusion.jpg" style="width:100%"/> | <img src="https://static.igem.org/mediawiki/2015/e/e2/Lambert_project_fusion.jpg" style="width:100%"/> | ||
<p>The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.</p> | <p>The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.</p> | ||
+ | |||
+ | <h2>Notebook </h2> | ||
+ | |||
+ | <h3> March</h3> | ||
+ | <p> Atlanta Science Festival </p> | ||
+ | <p> Received foldscope from Stanford Prakash Lab</p> | ||
+ | |||
+ | <h3> April </h3> | ||
+ | <p> Organized Chick Fil A Biscuit sale fundraiser </p> | ||
+ | <p> Worked with Next Generation Focus to provide science education enrichment </p> | ||
+ | |||
+ | <h3> May </h3> | ||
+ | <p> Trained new members in basic lab procedures </p> | ||
+ | |||
+ | <h3> June </h3> | ||
+ | <p> Researched yellow stripe rust/ CRISPR cas9 project idea </p> | ||
+ | <p> Researched PelB tags for relocating chitin deacetylase to the periplasm</p> | ||
+ | <p> Emailed FBI agents for outreach project</p> | ||
+ | <p> Researched expression vectors </p> | ||
+ | |||
+ | <h3> July </h3> | ||
+ | <p> Narrowed down focus of research to the PelB tags</p> | ||
+ | <p> Designed constructs using 3a assembly method </p> | ||
+ | <p> Designed constructs using gene synthesis method </p> | ||
+ | <p> Decided to use gene synthesis constructs due to resource restraints </p> | ||
+ | <p> Met with Georgia Tech iGEM team; received assistance for construct design </p> | ||
+ | |||
+ | |||
+ | <h3> August </h3> | ||
+ | <p> Completed constructs </p> | ||
+ | <p> Added a fusion protein </p> | ||
+ | <p> Practiced transformation procedures </p> | ||
+ | <p> Practiced digest procedures; fixed errors in procedure </p> | ||
+ | <p> Fixed illegal restriction sites in plasmids </p> | ||
+ | <p> Researched bidirectional and bicistronic constructs </p> | ||
+ | <p> Designed and received constructs from IDT </p> | ||
+ | <p> Resuspended sequences from IDT </p> | ||
+ | <p> Received filters from Rosco </p> | ||
+ | <p> Designed team shirts and jackets </p> | ||
+ | |||
+ | |||
+ | <h3> September </h3> | ||
+ | <p> Completed wiki </p> | ||
+ | <p> Completed banner </p> | ||
+ | <p> Submitted parts to parts registry </p> | ||
+ | <p> Designed handouts and flyers for Jamboree </p> | ||
+ | <p> Designed "Lab Hacks" outreach project for high school labs</p> | ||
+ | <p> Completed presentation for Jamporee </p> | ||
+ | <p> Completed poster for Jamboree </p> | ||
+ | |||
+ | |||
+ | <h2>Procedures </h2> | ||
</div> | </div> | ||
<div id="space2"> | <div id="space2"> |
Revision as of 23:36, 17 September 2015
Project
OVERVIEW
Lambert iGEM tackled this issue last year by dreaming up Chitinite, an inexpensive and bio-friendly alternative to current caustic and chemically intensive methods of chitosan production. This year we are continuing our project by further characterizing our CDA biobrick and expressing it in e.coli. Chitosan is toxic to e.coli, but by using a PelB tag to express CDA in the periplasm, we greatly reduce toxicity. This method facilitates cheap mass production of CDA. By making an eco-friendly fungicide accessible and cheap, Lambert iGEM hopes to protect the environment and help save the world.
Another highlight of our efforts was a Discovery Dialogue held in a partnership with the Atlanta Science Festival. We brought together on one stage, in an open forum; a legislator, ethicist, immunologist, FBI agent and scientist. The public was invited to ask questions and the concluding polls showed both an increase in the positive perception of synthetic biology, but also a request to hold another session this coming year.MATERIALS & METHODS
RESULTS
MODELS
PARTS
Main purpose of all the constructs:
Chitin deacetylase, CDA was isolated from S.cerevisiae. It is normally found in the ascopore wall during sporolation. We hypothesized that if we added a pelB localization tag to the CDA the protein would fold correctly in the periplasm. All of the constructs contain these genes: The pelB leader sequence, mature CDA2 gene, and the eGFP gene. The pelB leader sequence is necessary because it lead the CDA gene into the periplasm. The expression of the CDA gene in the periplasm will be non toxic to the E. coli cells. The eGFP will allow us to physically see that the DNA constructs were successfully transcribed and translated. The CDA protein produced will then be available to use to break down chitin and convert it into chitosan which has many industrial applications.
How to purify and use:
The constructs were ordered online at IDT and they came as gBlocks. (each construct contained 1000 ng). The constructs were resuspended as the instructions that came with the gBlocks stated. Then, they were digested (to eliminate the biobricks) and ligated into pSB1C3 and pSB1T3 plasmids. Once they were ligated into the plasmids, they were transformed into competent E. coli cells so that the genes may be expressed.
Construct 1: Bicistronic
The bicistronic construct consists of two promoters: the TetR promoter and the P(Lac) IQ promoter. This construct creates two different mRNA transcripts. One transcript will consist of the pelB leader sequence and the mature CDA2 gene which are both expressed under the TetR promoter while the other transcript will have the eGFP gene which is expressed under the P(Lac)IQ promoter. The eGFP will remain in the cytoplasm while the pelB leader sequence will lead the mature CDA2 gene into the periplasm. This means that the entire E. coli will glow green if the DNA construct is transcribed and translated correctly.
Construct 2: Bidirectional
The bidirectional construct only uses one promoter: the TetR promoter. This serves as an advantage because unlike the bicistronic construct, only one antibiotic (tetracycline) is needed to control gene expression. The construct puts two TetR promoters back to back with a multiple cloning site in between them. One TetR promoter controls the pelB + mature CDA2 genes while the other controls the eGFP gene. Once again, this creates two separate mRNA transcripts and the eGFP will be expressed in the cytoplasm while the mature CDA2 gene is led into the periplasm by the pelB leader sequence, just like the bicistronic construct.
Construct 3: Fusion
The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.
Notebook
March
Atlanta Science Festival
Received foldscope from Stanford Prakash Lab
April
Organized Chick Fil A Biscuit sale fundraiser
Worked with Next Generation Focus to provide science education enrichment
May
Trained new members in basic lab procedures
June
Researched yellow stripe rust/ CRISPR cas9 project idea
Researched PelB tags for relocating chitin deacetylase to the periplasm
Emailed FBI agents for outreach project
Researched expression vectors
July
Narrowed down focus of research to the PelB tags
Designed constructs using 3a assembly method
Designed constructs using gene synthesis method
Decided to use gene synthesis constructs due to resource restraints
Met with Georgia Tech iGEM team; received assistance for construct design
August
Completed constructs
Added a fusion protein
Practiced transformation procedures
Practiced digest procedures; fixed errors in procedure
Fixed illegal restriction sites in plasmids
Researched bidirectional and bicistronic constructs
Designed and received constructs from IDT
Resuspended sequences from IDT
Received filters from Rosco
Designed team shirts and jackets
September
Completed wiki
Completed banner
Submitted parts to parts registry
Designed handouts and flyers for Jamboree
Designed "Lab Hacks" outreach project for high school labs
Completed presentation for Jamporee
Completed poster for Jamboree