Team:NCKU Tainan/Measurement
Overview
An important aspect of Eye kNOw lies in ensuring that our bacteria will stay contained in the contact lenses. Bacteria biofilm has been shown to exhibit extraordinary ability to help bacteria itself bind to biotic and abiotic surfaces[1], which we exploited this property to design one of the biosafety measures.
Results
We engineered our bacteria to overexpress CsgD, a master transcription regulator of biofilm formation, and CsgA, the major subunit of curli fibers. Overexpression of these two proteins have been reported to increase biofilm formation[2], which we anticipated to help the bacteria bind to the contact lenses more securely, thus preventing the leakage of bacteria if the contact lens encounters any damage.
We first construct plasmid that only contains pLac-csgD and an improved version that contains both csgD and csgA under control of promoter T7 without LacO using gBlock provided by IDT. Furthermore, we used a common congo red staining protocol to characterize the biofilm amount. As shown in Fig. 1, there is an increase in congo red staining after 14 hours of culture.
However during experiments, we realized that most biofilm staining protocols used by researchers and other iGEM teams require steps to remove the unstained bacteria. These steps usually contain ambiguous descriptions such as “gently pipette the bacteria culture after staining”[3][4]. After several attempts at repeating these protocols, we soon discovered that most of the experimental errors came from the staining procedure. Also, common protocols testing biofilm formation only use staining dyes such as congo red[4] or crystal violet[3] to stain amyloid fiber curli fimbriae[5] or peptidoglycan[6], but these cannot directly support the claims of increasing binding affinity. Here, we developed a simple and quantifiable measurement protocol for assaying biofilm binding ability.
We first defined the increase in bacteria binding affinity as the ability to resist external forces applied to the bacteria culture.
According to the formula in Fig. 3a, the force a free-falling object can exert is proportional to its square root of the height, by elevating the object to different heights, we were able to modify the force exerted on our engineered bacteria precisely. Here, we utilize common accessible objects, in our case - a book. Since the book’s surface area is greater than the 96-well plate, it can exert the force evenly once it lands. As shown in the video below, we can simply drop the book from different heights and measure how well the bacteria can withstand those forces. Error bar indicates triplicate experiments
To measure the binding affinity of our engineered bacteria, we first grew the bacteria in a test tube until its OD600 value reached 0.4 and induced it with IPTG. Then, aliquot 200 μl of post-induction bacteria into the 96-well plate and incubate for 12 hours. After incubation, we take the 96-well plate and gently position it to our experiment setup. Right now, all the bacteria culture should stick to the bottom of the 96-well plate due to the surface tension. We then applied external forces to remove the excess bacteria culture, as biofilm forming colonies usually clump up around the culturing wells and are harder to remove[5]. We then use 200 μl ddH2O to resuspend and disrupt the remaining bacteria culture and measure the OD600 value to determine how much bacteria are retained after the force is applied.
We compared the binding ability of three bacteria previously characterized by congo red staining (BW25113, csgD, NOS-csgDA). As seen in Fig. 3, co-expressing csgD and csgA significantly increases the OD600 value when potential energy is applied compared to BW25113 and csgD alone, which congo red staining protocol only show minor difference between csgD and csgDA. Also, a great reduction in the standard deviation in each measurement is observed, suggesting that this protocol is accurate and reliable.
Thus, we are able to successfully establish a protocol that is not only simple to repeat, but is also able to support the claim of the enhanced binding ability of the engineered bacteria. Furthermore, our result shows that the congo red staining cannot reflect its binding ability. We highly recommend future iGEM teams to follow this protocol in order to explore the biophysical property of biofilm.
References
- DeBenedictis EP, Liu J, Keten S. Adhesion mechanisms of curli subunit CsgA to abiotic surfaces. Science Advances. 2016;2(11):e1600998.
- Brombacher E, Baratto A, Dorel C, Landini P. Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion. Journal of Bacteriology. 2006;188(6):2027-2037.
- Merritt JH, Kadouri DE, O’Toole GA. Growing and Analyzing Static Biofilms. Current Protocols in Microbiology. July 2005.
- Team:TAS Taipei - 2017.igem.org. 2017.igem.org. https://2017.igem.org/Team:TAS_Taipei. Accessed September 27, 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.
- Petrachi T, Resca E, Piccinno M, et al. An Alternative Approach to Investigate Biofilm in Medical Devices: A Feasibility Study. International Journal of Environmental Research and Public Health. 2017;14(12):1587.
