Team:Harvard BioDesign/Project

Project description

Overall 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.
            We designed a sensing system in E. coli to store information about the cells’ environment by linking curli proteins to half of a strong affinity tag pairing. The two peptides that compose an affinity tag pair bind only to each other, allowing information to be stored until both peptides are present. Using a variety of affinity tags, we placed each one under the control of a different inducible promoter. The ratio of different affinity-tagged curli produced by the cells is determined by the relative concentrations of the inducers present in the environment. Next, we created fusion chromoproteins with the orthogonal affinity tags. By applying a standard mixture of affinity-tagged chromoproteins, we identify the ratio of bound chromoproteins via an RYB color system. This ratio corresponds to the information encoded by the E. coli. This work has potential applications in the fields of cryptography, information storage, as well as the formation of a new reporting toolbox for synthetic biology.

Project Details
Our system is composed of the following components:

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
Genetic constructs were created by PCR cloning reactions, Gibson assembly reactions, and related protocols. CsgA production was monitored via Congo Red assays, and chromoprotein-CsgA association was determined by culturing both transgenic proteins together for 24 hours, subjecting the solution to SDS gel electrophoresis, and observing the resultant protein bands for conjugation between the SpyTag and SpyCatcher-associated proteins.

The Experiments

• Congo red assays
Congo red is a secondary diazo dye with a high affinity to amyloid fibers. Congo red spin down assays were used to quantify curli fiber productio. 100uL of a .015% solution of Congo red is incubated with 1mL of LSR10 cells producing csgA constructs resuspended in PBS. LSR10 cells have cellulose production knocked out as not to interfere with the specificity of congo red binding. Cells are pelleted and supernatant transferred to a 96-well plate to measure absorbance at 490nm. Cells that express more CsgA pull down more congo red from the supernatant resulting in lower absorbance levels.
• Gel shift assays
Chromoprotein engineered with spycatcher (mRFP-Spycatcher) was expressed in LSR10 cells, lysed, and purified using a Ni-NTA column. Cells producing WT-CsgA and CsgA-Spytag were sheared using a sonication probe and spun down to isolate CsgA in supernatant. Supernatant was incubated with crude cell lysate and purified mRFP-Spycatcher over night then run on an SDS-Page gel. We expect part of the mRFP-Spycatcher population to shift up when bound to CsgA-Spytag.
• Colormetric assays
Chromoproteins obtained from Uppsala 2011 were mixed at different ratios to indicate that a range of distinguishable color gradients can be achieved to detect relative levels of input by eye using a chromoprotein-binding schematic with multiple affinities.

Results

• Gel Shifts
According to the results we have obtained thus far, modifying native CsgA to include our selected binding domain reduces the total amount of curli that is ultimately produced by E. Coli (according to Congo Red analysis), by anywhere from 20-50%. However, the curli that is produced is stained by Congo Red, indicating that it has chemical similarity to the curli produced by wild-type cells.

• Congo red assays

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.


• Colormetric assays

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
The reduction in curli production that was observed after we added binding domains to CsgA is not too great to make our system unworkable. Various methods can be used to counteract this problem, the simplest of which is to simply use a greater initial number of cells such that the desired or required amount of curli is ultimately produced. In the future, we may attempt to determine whether using linker sequences with different lengths, compositions, and 3-dimensional structures can affect the amount of curli that is ultimately produced – it is possible that the shape of the modified CsgA subunit is less optimal for the self-assembly of curli that is an important feature of our system.

Conclusion
The results of our Congo Red analysis indicate that it is possible to produce significant amounts of curli with E. coli engineered to express mutant variants of CsgA with binding domains that allow our data processing and storage system to function. Small adjustments will be made to optimize curli production and self-assembly, but we have shown that it is possible to modify CsgA in ways required for our system to function without limiting curli production to an unacceptable degree.