Team:BostonU/FusionProteins

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As described <a href="https://2014.igem.org/Team:BostonU/Chimera"> here</a>, 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 <a href="https://2014.igem.org/Team:BostonU/Workflow"> workflow</a>, we started designed and started building a <a href="https://2014.igem.org/Team:BostonU/Encoder"> priority encoder</a>. To facilitate measurement of each component, we added the fluorescent fusion proteins to the priority encoder design. Since we lacked a lot of the internal repressor genes that we needed, we decided to test well known proteins available in our lab (<i>tetR</i>, <i>lacI</i>, and <i>araC</i>) with a GFP fusion protein before using this linker sequence for all fusions.<br><br>

<center><img src="https://static.igem.org/mediawiki/2014/b/be/BU14_PE_Updated_fromPP.png" width="65%"></center>

A peculiar effect for these types of designs has been reported in the literature. Specifically, 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 genes further down a transcript are expressed less than the genes upstream [1]. <br><br>

<center><img src="https://static.igem.org/mediawiki/2014/9/9a/BU2014_Fig_4.jpg" width="40%"</center>

<capt><br><br><center>The image above is Figure 4 from Lim et al., 2011 [1]. Excerpt from Lim N H et al. (2011): "We found that CFP and YFP expression at the first position in the operons was higher than at the third position for each time point. The protein concentration at each time point was normalized by the final concentration to determine their relative expression (R). R is independent of protein production and depends only on the protein degradation rate and any delay in the induction (SI Materials and Methods). When R was calculated, it was the same for positions 1 and 3 at each time point (Fig. 4 D and E). Therefore, protein degradation and any delay after induction are the same at both positions, indicating the differences in expression must be due to greater protein production in the more proximal position of the operons."</center></capt></center>

 

<br><br>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]. Since these constructs are designed to measure protein degradation rate directly, these types of designs are inefficient. This problem can be solved by fusing the regulator protein with the fluorescent protein, as only one ribosome will then be required to translate the entire sequence, eliminating this problem.  

 

Another way to measure function by fluorescence, using consecutive transcriptional units

Because it takes a minimum of 3 days to build these types of devices using MoClo these types of designs result in the expenditure of more time and resources as compared to building single transcriptional units with fusion proteins. Any design involving just one transcriptional unit instead of the above construct will making cloning faster and more cost-effective. <br><br>

Fusion Proteins are fused to regulator proteins to allow us to measure degradation rate directly. They reduce order effect seen with the bicistronic design and are cheaper to build, compared to cloning multiple TUs.<br>

<h3>Design and Assembly</h3>

To make fusion proteins, we used the <a href="https://2014.igem.org/Team:BostonU/MoClo"> Modular Cloning </a>method .  

First, we added a new MoClo fusion site (I - TCTA) to the genes (at the end of repressors and before the reporter proteins). The designed primer sequences can be accessed on the<a href="https://2014.igem.org/Team:BostonU/Data"> Data Collected</a> page.

The following Level 2 constructs were assembled using MoClo -

<center><img src="https://static.igem.org/mediawiki/2014/d/da/YAFP6BUWiki.png" width="700" style="float:center"></center>

<center><img src="https://static.igem.org/mediawiki/2014/2/23/YAFP7BUWiki.png" width="700" style="float:center"></center>

Experimental Results can be found in the <a href="https://2014.igem.org/Team:BostonU/Data"> Data Collected </a>page.

[1] Lim N H, Lee Y, Hussein R (2011) "Fundamental relationship between operon organization and gene expression" ,  PNAS Vol. 108 No. 26, doi: 10.1073/pnas.1105692108

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