Template:Lambert GAprojects
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.
Our project also highlights the Foldscope, a paper microscope developed by the Stanford Prakash Lab. As a high school team, we struggled firsthand with the lack of resources and lab equipment that plagues many other high school iGEM teams. The foldscope is a cheap and effective microscope with a wide range of uses. By investigating applications of the Foldscope in a bioengineering context, we hope to allow more high school teams without funds for expensive lab equipment to pursue their passions in synthetic biology.MATERIALS & METHODS
RESULTS
MODELS
PARTS
Main purpose of all the constructs:
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.
