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	<p align="center">Fig.4. The binding affinity of wild type, CsgD, and CsgA-CsgD at different heights of force. The two asterisks represent 0.001 value, and three asterisks denote 0.0001 value.  </p>		<p align="center">Fig.4. The binding affinity of wild type, CsgD, and CsgA-CsgD at different heights of force. The two asterisks represent 0.001 value, and three asterisks denote 0.0001 value.  </p>

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Latest revision as of 21:12, 27 October 2020

CsgD, the master regulator of biofilm formation

CsgD, the master regulator of biofilm formation, activates the synthesis of curli fimbriae and extracellular polysaccharides in Escherichia coli.

Characterization and Improving Function

Team: TAS_Taipei 2017

Author: Justin Yang

SDS-PAGE

BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size. SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively. Predicted proteins from the curli operons are listed on the right, and E. coli expressing GFP was used as a positive control.

CONGO RED ASSAY

We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips, and incubated at 37˚C for one day. If biofilms were present, the solution would appear red, which could be quantified by an absorbance value at 500 nm. Overexpression of CsgD (BBa_K2229100) in our experiments doubles biofilm production compared to the control BBa_K805015. When bacteria expressing CsgD were plated in petri dishes, biofilms appeared thicker compared to controls.

Overexpression of CsgD (BBa_K2229100) doubles biofilm production. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

BBa_K2229300 Improves the Function of BBa_K805015

Team: TAS_Taipei 2017

Author: Justin Yang

Our new composite part BBa_K2229300 improves the function of two existing parts: BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF). CsgD and OmpR234 are regulators of two curli operons, which contribute to biofilm formation. When both proteins are overexpressed, we hypothesized that twice the amount of curli monomers should be made and exported to form fibers and biofilm.

SDS-PAGE

Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) showed two extra bands at 15 kDa and 30 kDa, which were not observed in cultures expressing either CsgD or OmpR234 alone. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012). This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in the SDS-PAGE figure above).

CONGO RED ASSAY

Overexpression of both CsgD and OmpR234 (BBa_K2229300) increases biofilm production the most. A) Congo red assay stains biofilms. BBa_K2229300 increases adhesion to glass surfaces. B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

When all three expression constructs were compared, we find that overexpression of OmpR234 and CsgD together (BBa_K2229300) increased biofilm production the most. BBa_K2229300 also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps.

BBa_K3490001 Improves the Function of BBa_K805015

Team: NCKU_Tainan 2020

Author: Virginia Clarence Setiawan

Improvement: 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.^[2] As a transcription factor of curli proteins, CsgD can regulate the expression of csgA, leading to biofilm production.
Therefore, we are trying to improve BBa_K805015 from the 2017 iGEM TAS-Taipei Team^[3] to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lenses 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 can initiate the bacteria to bind securely to the contact lenses. 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

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 is increasing, we did a test using congo red^[4] 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 increases 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 being produced by bacteria with different genetic backgrounds at different times.

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 conduct another experiment to compare the binding affinity of bacteria among control, csgD, and csgA-csgD. We presume that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we threw 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. The binding affinity of wild type, CsgD, and CsgA-CsgD at different heights of force. The two asterisks represent 0.001 value, and three asterisks denote 0.0001 value.

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 BBa_K3490001.

Reference

[1] BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org. https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM. Published 2020. Accessed September 9, 2020.
[2] Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
[3] Part:BBa K805015 - parts.igem.org. Igem.org. https://parts.igem.org/Part:BBa_K805015. Published 2013. Accessed September 21, 2020.
[4] 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.

Sequence and Features