Team:Dalton School NY/Project

From 2012hs.igem.org

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. Protein-coding sequences are the parts of genes that actually code for proteins. Terminators are the parts of genes that stop transcription. The goal of the Johns Hopkins Build-A-Genome project is to create libraries of “parts” that can be mixed-and-matched by means of combining any of a plethora of combinations of promoters, protein-coding sequences, and terminators. This enables an efficient method of creating a diverse array of genes with equally diverse functions. Our iGEM here at the Dalton School has taken on a small portion of this Hopkins project. The Dalton Team is responsible for putting 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 successful and efficient.We added certain primers to the DNA extracted from this trial to target specific genes to amplify. 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 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 in order to create our final product.

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 are currently being 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. We plan to sequence positive clones. We initially cloned 25 of 30 promoters and are currently working on obtaining the other five in addition to the six fluorescent protein genes.

Creating libraries of gene parts:

1. Use PCR to copy promoters, protein-sequences, and terminators. The PCR primers have tails that contain BsaI sites.

PCR of a promoter from genomic DNA:

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Final PCR products, showing the sequence added to the ends by the primer tails (not to scale):

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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.

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Assembling gene parts into functional genes:

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:

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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.

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