Team:Harvard BioDesign/Project
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<li>Congo red assays</li> | <li>Congo red assays</li> | ||
- | Congo red spin down data suggests that there is some curli fiber production in engineered constructs, however, production is much lower compared to wild type CsgA. | + | <p>Congo red spin down data suggests that there is some curli fiber production in engineered constructs, however, production is much lower compared to wild type CsgA. </p> |
<img src="https://static.igem.org/mediawiki/2014/e/e6/HarvardCongoRedData.jpg" width="800px" height = "450px"/> | <img src="https://static.igem.org/mediawiki/2014/e/e6/HarvardCongoRedData.jpg" width="800px" height = "450px"/> | ||
<li>Colormetric assays</li> | <li>Colormetric assays</li> | ||
- | mRFP (BBa_E1010), aeBlue (BBa_K1033929), and amilGFP(BBa_K1033931) from team Uppsala 2011 were mixed at ratios of 10:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:10 to obtain color gradients. | + | <p>mRFP (BBa_E1010), aeBlue (BBa_K1033929), and amilGFP(BBa_K1033931) from team Uppsala 2011 were mixed at ratios of 10:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:10 to obtain color gradients. </p> |
<img src="https://static.igem.org/mediawiki/2014/4/4e/Rainboli.JPG" width = "600px" height="450px"/> | <img src="https://static.igem.org/mediawiki/2014/4/4e/Rainboli.JPG" width = "600px" height="450px"/> | ||
Latest revision as of 01:49, 18 October 2014
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Project descriptionOverall Project Summary E. coli, along with many other gram-negative bacteria, produce beta-amyloid proteins called curli which form the basis of their biofilms. These amyloid structures are highly resistant to degradation and can survive extreme pH and temperature changes. Such robust features of curli proteins make them a great medium for the stable encoding of information. Additionally, curli fibers are assembled extracellularly throughout the lifetime of the cells, so information stored in curli can be read well after cells have died. Project Details CsgA variants - We have engineered four versions of the CsgA protein with four different affinity domains: three coiled coil SynZip domains, and one covalent bond-based SpyTag domain. The affinity domains are fused to the endogenous CsgA with a 24-amino acid linker sequence, to ensure that the domains are available for binding as the CsgA polymerizes into a curli fiber. Chromoprotein constructs - to demonstrate the feasibility of our concept, we used chromoproteins fused to binding domains reciprocal to those fused to the CsgA variants. These are three SynZip domains, and one SpyCatcher domain, which spontaneously forms a covalent bond with the SpyTag domain when the two come close enough to one another in solution. Sensing promoters – thus far we have coupled expression of the CsgA variants to one inducuble promoter: LacZ. In the future we hope to include promoters induced by more interesting environmental conditions, like temperature or atmospheric chemical concentrations (i.e. carbon monoxide), so that the composition of the curli fibers produced by the bacteria will reflect such environmental conditions. Materials and Methods The Experiments
Results
Congo red spin down data suggests that there is some curli fiber production in engineered constructs, however, production is much lower compared to wild type CsgA. mRFP (BBa_E1010), aeBlue (BBa_K1033929), and amilGFP(BBa_K1033931) from team Uppsala 2011 were mixed at ratios of 10:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:10 to obtain color gradients. Analysis Conclusion |