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Team:Dalton School NY
From 2012hs.igem.org
Our team is composed of 5 juniors in the Advanced Biotechnology and Molecular Biology class at The Dalton School in New York, NY. We are collaborating on the yeast Build-A-Genome project at Johns Hopkins University.
Contents |
Introduction
Students in the Advanced Biotechnology and Molecular Biology course at The Dalton School are collaborating with scientists and students at Johns Hopkins University on their effort to construct a synthetic yeast genome. The short term-goals of this project are to construct large libraries of promoters, protein-coding sequences, and terminators that can be easily combined to form functional genes. Dalton students are cloning 30 promoters as part of this effort. In addition, we are also cloning the protein-coding sequences for 6 fluorescent proteins that can eventually be combined with the promoters to test the strength of the promoters. After constructing genes, the individual genes can be assembled into synthetic yeast chromosomes ([http://www.nature.com/nature/journal/v477/n7365/full/nature10403.html read the Nature paper]). The long-term goal of the Hopkins Build-A-Genome initiative is to be able to engineer yeast that can be used to solve human problems including combating world hunger, producing alternative sources of fuel, and studying human disease pathways in a simplified system. For example, last year, Hopkins students inserted all of the enzymes necessary to produce vitamin A into yeast. These yeast can be used to bake bread containing vitamin A to supplement the diets of malnourished people. Please visit Johns Hopkins iGEM 2011.
Team
Meet our team members!
Project
The wildtype yeast genome contains about 6000 genes on 16 chromosomes. In order to accomplish the goal of creating libraries of gene parts, thousands of different promoters, protein-coding sequences, and terminators need to be cloned into individual plasmids. Promoters are the parts of genes that initiate transcription and contain the 5'UTR of the mRNA. Protein-coding sequences are the parts of genes that actually code for proteins. Terminators are the parts of genes that stop transcription and contain the 3' UTR of the mRNA.
The goal of the Johns Hopkins Build-A-Genome project is to create libraries of gene parts that can be mixed-and-matched by means of combining individual promoters, protein-coding sequences, and terminators. This enables an efficient method of creating a diverse array of genes with equally diverse functions. Our iGEM team here at the Dalton School has taken on a small portion of this Hopkins project. The Dalton iGEM Team is responsible for cloning 30 different promoters into individual plasmids (pUC19) as well as 6 different protein-coding sequences. The protein-coding genes we are cloning include 6 different fluorescent protein genes derived from jellyfish and coral that have been developed in Roger Tsien’s lab. These fluorescent protein genes will allow us and the Hopkins students to characterize the strength of the yeast promoters that we clone by monitoring the fluorescence of yeast containing a promoter linked to a fluorescent protein-coding sequence.
We began this experiment by testing different methods of purifying DNA from the yeast genome. Based on the results from the gel we ran with DNA purified via multiple methods, the Promega method was the most consistenly reliable in our hands.
Creating libraries of gene parts:
After obtaining genomic DNA, we used this DNA as the template in PCR reactions using primers that amplified individual promoters. Each of these primers include tails that have the recognition site for the restriction enzyme BsaI. This is necessary because BsaI is a unique enzyme that doesn’t cut at it’s recognition site, but instead cuts a few bases down, thus cutting off its own recognition site. Therefore, every time it cuts, BsaI creates sticky ends that will be complimentary to whichever gene part succeeds it. For example, the few nucleotides following the promoter will be complimentary to the few nucleotides preceding the protein-coding sequence. This is one of the coolest and most distinct parts of the Hopkins project; it allows us to put a promoter, a protein coding sequence, a terminator, and only one restriction enzyme (BsaI) into a tube with ligase in order to create our final product.
1. Use PCR to copy promoters, protein-sequences, and terminators. PCR primers were ordered from IDT. The PCR primers have tails that contain BsaI sites.
PCR of a promoter from genomic DNA:
Final PCR products, showing the sequence added to the ends by the primer tails (not to scale):
After PCR, we ran our samples on a gel and cut out the band of the correct size. We then purified the DNA from the gel by means of a gel extraction. Next, we cloned our PCR products into the pUC19 vector. We used the restriction enzyme SmaI to digest the pUC19 vector, which creates blunt ends that need to be dephosphorylated to prevent religation. This digestion of pUC19 allows us to ligate in our PCR product to create the complete plasmid. This is the final product that we will contribute to the Hopkins collection. The ligated vectors were transformed into E.coli. Next, we picked colonies from the transformation. After this we mini-prepped the DNA from these colonies and confirmed that the bacteria contained the correct inserts by performing BsaI digests. BsaI cut out the inserted DNA for any correct clones. We plan to sequence positive clones. We initially cloned 25 of 30 promoters and have recently obtained the other five in addition to the six fluorescent protein genes.
2. Blunt-end clone the PCR products into a pUC19 plasmid that has been digested with SmaI. This creates a library of modular gene parts that are flanked by BsaI sites.
Assembling gene parts into functional genes:
(We haven't tried this yet, but hope to do so soon).
1. Mix 3 pUC19 vectors containing one promoter, one protein-coding sequence, and one terminator. Also add a yeast cloning vector containing BsaI cut sites.
2. Add BsaI restriction enzyme which will release parts with the following overhangs:
3. Add DNA ligase which will paste together fragments with complementary overhangs. Note that there is no need to remove the BsaI restriction enyme before this step because it cuts off its own recognition site, so the final desired product cannot be cut with BsaI. This mixture of ligated final plasmids and other DNA fragments can be transformed into E. coli and only the circular, final plasmid will allow colonies to grow.
Notebook
Please view our lab notebook.
Results/Conclusions
Please view our results/conclusions.
Safety
Please click here to learn how we adopted protocols that are safe for a high school classroom.
Sponsors
We are very appreciative of our sponsors - this work would not have been possible without them!
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