Team:BostonU/FusionProteins

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Revision as of 00:17, 24 July 2014

Fusion Proteins

Need for Fusion Proteins

Fusion Proteins are fused coding sequences that allow us to use one transcriptional unit (with a repressor and reporter protein fused together) rather than two units (one with the repressor and the other with the reporter protein, whose expression depends on the expresser of the repressor).

Fusion Proteins play a very important part in Project Chimera. In addition to helping us reduce the total transcriptional units used, they also allow for greater expression of tandem coding sequences.

If we look at constructs that have repeating sequences of ribosome binding sites and reporter proteins in one transcriptional unit, a peculiar effect can be observed. When ribosomes bind to the genes sequentially, the ribosome that binds before might block the next ribosome from binding to the mRNA and translating the gene. So, the later genes are expressed less than the genes before. This problem is solved with fused proteins as only one ribosome will then be required to translate the entire sequence, eliminating any possible problems during translation.

Making Fusion Proteins

To make fusion proteins, we used the Modular Cloning method that we have used for most digestion-ligation reactions.

First, we added a new MoClo fusion site (I - TCTA) to the genes (at the end of repressors and before the reporter proteins). The fusion site, I (TCTA) was then added to another 2-nucleotide sequence (GA) to make the XbaI site. This was done to allow the two proteins made by the two gene to split up after translation.

Phusion Polymerase Chain reaction was performed on the basic MoClo Level 0 parts using primers designed based on the fusion sites. We ended up with repressors with TCTAGA sequence at their ends and reporter proteins with the same sequence at the start. These can be treated as standard Level 0 parts which can then be assembled and tested using MoClo.

Testing Fusion Proteins

Fusion Proteins are fused coding sequences that allow us to use one transcriptional unit (with a repressor and reporter protein fused together) rather than two units (one with the repressor and the other with the reporter protein, whose expression depends on the expresser of the repressor).

Fusion Proteins play a very important part in Project Chimera. In addition to helping us reduce the total transcriptional units used, they also allow for greater expression of tandem coding sequences.

If we look at constructs that have repeating sequences of ribosome binding sites and reporter proteins in one transcriptional unit, a peculiar effect can be observed. When ribosomes bind to the genes sequentially, the ribosome that binds before might block the next ribosome from binding to the mRNA and translating the gene. So, the later genes are expressed less than the genes before. This problem is solved with fused proteins as only one ribosome will then be required to translate the entire sequence, eliminating any possible problems during translation.

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-Fusion Proteins are fused coding sequences that allow us to use one transcriptional unit (with a repressor and reporter protein fused together) rather than two units (one with the repressor and the other with the reporter protein, whose expression depends on the expresser of the repressor).
+To make fusion proteins, we used the Modular Cloning method that we have used for most digestion-ligation reactions.
 <br><br>
-Fusion Proteins play a very important part in Project Chimera. In addition to helping us reduce the total transcriptional units used, they also allow for greater expression of tandem coding sequences.
+First, we added a new MoClo fusion site (I - TCTA) to the genes (at the end of repressors and before the reporter proteins). The fusion site,  I (TCTA) was then added to another 2-nucleotide sequence (GA) to make the XbaI site. This was done to allow the two proteins made by the two gene to split up after translation.
 <br><br>
-If we look at constructs that have repeating sequences of ribosome binding sites and reporter proteins in one transcriptional unit, a peculiar effect can be observed. When ribosomes bind to the genes sequentially, the ribosome that binds before might block the next ribosome from binding to the mRNA and translating the gene. So, the later genes are expressed less than the genes before. This problem is solved with fused proteins as only one ribosome will then be required to translate the entire sequence, eliminating any possible problems during translation.
+Phusion Polymerase Chain reaction was performed on the basic MoClo Level 0 parts using primers designed based on the fusion sites. We ended up with repressors with TCTAGA sequence at their ends and reporter proteins with the same sequence at the start. These can be treated as standard Level 0 parts which can then be assembled and tested using MoClo.
 <br><br><br>
 <h3>Testing Fusion Proteins </h3>