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Team:BostonU/FusionProteins
From 2014.igem.org
Why Fusion Proteins?As described here, our main goal this summer was to come up with an efficient workflow to design, build and test large genetic devices. To demonstrate the efficacy of our workflow, we started building a priority encoder. Below, we added the fusion proteins to mark where we will need them for the priority encoder. However, when we began we lacked a lot of the internal repressor genes that we wanted to use, so we decided to test well known proteins available in our lab (tetR, lacI, and araC) with a GFP fusion protein. In order to test the individual transcriptional units and the regulatory arcs associated with them, we needed the output for the individual parts of the device to be fluorescent. One way to do this is by using the bicistronic design shown below that mimics operons seen in natural bacterial systems.  A peculiar effect was observed in relation to this design. When ribosomes bind to the genes sequentially, the ribosome that binds at the 5' end of gene 1 can interrupt the attachment of a ribosome at the 5' end of gene 2, thus blocking the next ribosome from binding to the mRNA and translating the gene. So, the later genes are expressed less than the genes before [1]. 
In summary, when genes are placed in this operon strategy in synthetic systems, the first gene always expresses higher amounts of protein than the second or third genes [1]. The whole point of these constructs is to measure regulator degradation rate directly. So, these are rendered inefficient if the reporter and regulator don't have comparable expression. This problem can be solved by fusing together two proteins as only one ribosome will then be required to translate the entire sequence, eliminating any possible problems during translation. Another way to measure function by fluorescence is by using consecutive transcriptional units -  It takes this 6 days to build these devices using BioBricks and 3 using MoClo. This clearly results in the expenditure of more time and resources as compared to building simple transcriptional units. Any design involving just one transcriptional unit instead of the above construct will making cloning faster and more cost-effective. Fusion Proteins are fused coding sequences that allow us to measure degradation rate directly. They reduce order effect seen with the bicistronic design and also, should theoretically be much easier to build as compared to cloning multiple TUs.  Design and Assembly 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 genes to split up after translation. Then, we used Phusion Polymerase Chain reaction 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. TestingIt is important that the fusion proteins made aren’t drastically inferior to the individual action of the repressor or the fusion proteins. In order to test this, the following Level 2 constructs were assembled using MoClo -   Here onwards, all constructs made were to compare with those above. Simply put, all 6 Level 2s below were used a controls. KEY
Detailed progress on the construction of fusion proteins can be found in the Fusion Proteins notebook.
[1] Lim N H et al. (2011) "Fundamental relationship between operon organization and gene expression" , PNAS Vol. 108 No. 26, doi: 10.1073/pnas.1105692108
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