Improvement

Practice makes perfect

Overview

One of the goals we want to achieve is to attach our bacteria to the inner chamber of the contact lens. Here, we used csgD, a master regulator of biofilm production; and csgA, a major subunit of the curli fimbriae. Based on research, curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation^[1]. As a transcription factor of curli proteins, csgD can regulate the expression of csgA, leading to biofilm production.

Therefore, we are trying to improve BioBrick BBa_K805015 from the 2017 iGEM TAS-Taipei Team^[2] to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lens despite any damages.

We hypothesized that by adding csgA and pLac to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm enables the bacteria to bind securely to the contact lens. Therefore, we add csgA to the biobrick design and change the promoter into pLac as an improvement.

Fig. 1. A schematic of our biobrick construction.

Experiment Results

Congo Red Assay Experiment

First, we ran SDS-PAGE to identify and quantify the protein expression of CsgA and CsgD. We cultured the bacteria for 2 hours, then added IPTG to induce for 12 hours long, and adjusted the OD₆₀₀ value to three. As a comparison, we used plasmid that contains pLac-csgD on BW25113, improved parts that include NOS, csgA, and csgD on BW25113, and using PCA24N as control. After that, we transformed into E. coli BL21(DE3) strain. The expected protein size of CsgD is around 24 kDa and CsgA around 17 kDa. The results below have shown the outcome we expected for CsgA and CsgD protein expression.

Fig. 2. SDS-PAGE of E.coli BL21(DE3). M: Marker; Lane 1: Wild type CsgD in PCA24N; Lane 2: Wild type BW25113 (control); Lane 3: Knockout CsgD in PCA24N; Lane 4: Knockout BW25113 (control); Lane 5: BL21(DE3)-NOS-CsgA-CsgD. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).

Next, to prove that our amount of biofilm production increased, we did a test by using congo red^[3] dye to observe the curli expression. We compared the absorbance value of BBa_K805015 and BBa_K3490001 to see whether the amount of biofilm production increased or not. If the biofilm amount increases, the color of the solution will appear to be darker. So, after overnight culture, we add congo red dye to all the samples. Then we centrifuge to separate the supernatant and precipitate (pellet). By using a microplate reader, we can measure the absorbance value at 500 nm (congo red) and normalized by 600 nm wavelength which represents the amount of bacteria.

Fig. 3. Amount of biofilm produced by bacteria with different genetic backgrounds at different times.

Biofilm Assay Experiment

However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we conducted another experiment to compare the binding affinity of bacteria among control, csgD, and csgA-csgD. We presumed that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we dropped a book from different heights and measuring its OD₆₀₀ value. By doing so, we can determine the concentration that represents the binding affinity.

Fig. 4. (A) The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.) (B) The binding affinity of control, *csgD*, and *csgA-csgD* with given force from different heights.

As seen in the graph above, csgA-csgD shows an increase in OD₆₀₀ value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick (BBa_K805015) by adding csgA and changing the promoter into pLac (BBa_K3490001).
Please visit Results page for more information.

References

Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
Part:BBa K805015 - parts.igem.org. Igem.org. http://parts.igem.org/Part:BBa_K805015. Published 2013. Accessed September 21, 2020.
Jones CJ, Wozniak DJ. Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. c-di-GMP Signaling. 2017:147-156.