Experimental Overview
To engineer bacteria with the capacity to produce and regulate biofilm, we utilized the functions of the curli operon and the metJ regulon, respectively, to create two constructs. Additionally, to address the risk of unwanted contamination by our engineered bacteria, we have also designed an toxin-antitoxin system (inspired by the Johns Hopkins 2021 team).
To assess the functionality of these engineered bacteria and the impact of our biofilm-related constructs, we have devised three key assays to evaluate the functionality of our engineered bacteria: a biofilm production assay, a MetJ assay, and a biosafety assay.
We performed multiple cycles to assemble our three desired plasmids, we will be using the pSB1C3 for our biofilm-producing construct (Csg construct), pET28a for the biofilm-regulating construct (MetJ construct), and the plasmid pET21a for Biosafety construct.
Csg Construct[1]
Figure 1.a A diagram of our Csg construct
Our Csg construct is responsible for producing and regulating biofilm (together with the MetJ construct). We designed our construct on benchling, added biobrick prefix and suffix, and provided its sequence to IDT for synthesis; the synthesized product was amplified via PCR and ligated with pSB1C3 via biobrick assembly. The assembled plasmid was transformed into E. coli DH5Alpha, miniprepped, and sequenced for confirmation.
For cloning results, please visit the engineering page
During the design process, we conducted several literature reviews and found that csg genes are responsible for biofilm production in E. coli, which are commonly used and studied, and known for its highly resilient and adaptive qualities — which are crucial to survival in ocean environments. Its non-pathogenic qualities also are key to our selection. These genes encode for the major component of curli, which is a proteinaceous fiber that forms the structural backbone of the biofilm. By engineering the csg genes into SAR11 bacteria, it allows SAR11 to produce biofilm and facilitate its integration into the coral microbiome. Research was done to challenge certain strains of viruses containing csgF and csgG, cell lysis mechanisms that would destroy the cell membrane, in SAR11. However, it was proved that the virus didn’t infect any of the SAR11, confirming that Csg doesn’t affect SAR11 [2]. Additionally, it is also proven that SAR11 can allow the production of biofilm. Research showed that SAR11 was found in the RO biofilm communities, suggesting SAR11 cells are present within the biofilm structure, confirming SAR11's capacity to facilitate the production of biofilm, along with the production of probiotics [3].
Figure 2. Full animation of the Csg construct
The csg operon (curli operon)[1]
In order to achieve this, we synthesized the csg genes found in E. coli MG1655 and inserted it into our Biofilm construct. We have only selected parts of the csg genes, which include csgB, csgA, csgC, csgE, csgF and csgG. This selection of csg genes from E. coli MG1655 was based on their known role in curli fiber expression, secretion, and biofilm [4].
The csgB gene initiates the process of curli fiber assembly by acting as a nucleator for the polymerization of CsgA, the major curli subunit. Meanwhile, CsgC protein acts as a chaperone that prevents the premature polymerization for CsgA. The csgE and csgF genes interact with the csgG gene to increase the efficiency of curli assembly.
T7 promoter
By using T7 promoter, we can achieve high levels of expression of csg, which is responsible for curli fiber production and biofilm formation in our SAR11 bacteria.
Figure 3. Animation of T7 promoter in Csg construct
Figure 4. Animation of met operator in Csg construct
MetJ Construct
Figure 1.b A diagram of our MetJ construct
Our MetJ construct is responsible for regulating biofilm. The construct sequence was designed using benchling and then sent to IDT for synthesis. After receiving our synthesized construct, we amplified and added complementary basepairs to pET28a and performed Gibson assembly. This assembled plasmid was subsequently transformed into E. coli DH5Alpha cells, followed by miniprep purification and sequencing analysis to confirm the accuracy of the construct's sequence.
For cloning results, please visit the engineering page
Met Operator
We also employed the regulatory protein MetJ to suppress biofilm production to mitigate the suffocation of corals from the overgrowth of biofilm, and to aid in that process we have integrated a met operator into our construct. By binding to the met operator, MetJ effectively reduces the synthesis of biofilm.
The Met Regulon
Due to initially rapid production of biofilm required for the attachment of our engineered bacteria, the intended probiotic, and the coral mucosal layer, we decided to implement T7 promoter, which acts as a strong constitutive promoter for the production of biofilm. However, this opens the door to a variety of problems.
The most glaring of these problems is the possibility of disease. Biofilm acts as an ideal environment for not only the bacteria that we intend to use as part of our solution, but also to various pathogens that can develop on the biofilm. The accumulation of the pathogenic bacteria on the biofilm increases rates of coral disease [5]. The most outstanding example of this would be the possibility of it serving as a reservoir for pathogens that causes the Stony Coral Tissue Loss Disease [6].
Another problem that we face with biofilm is the blocking of sunlight to the coral. Corals are dependent on a species of algae called zooxanthellae for the production of their food through photosynthesis. In some species, up to 90% of their consumption could be dependent on zooxanthellae [7]. Consequently, the blocking on sunlight toward coral from biofilm may affect the coral’s health.
To address these issues, we utilized the MetJ regulatory protein in a quorum sensing mechanism, a regulatory protein that represses the production of biofilm by attaching to the met operator. As the bacterial population increases over time, so does the concentration of the autoinducer (AHL). Autoinducers are signaling molecules that are produced in response to changes in cell-population density. As the density of quorum sensing bacterial cells increases so does the concentration of the autoinducer [8]. Under conditions of low population density, there will be a low concentration of AHL, resulting in the low production of MetJ proteins. Due to the low binding affinity of MetJ, the production of biofilm under these conditions will be high. Under high population density, the high AHL concentration will induce the production of MetJ, which will then decrease biofilm production, allowing the effective control of the gene expression responsible for biofilm formation.
Figure 5. Full animation of MetJ construct
CinR-CinI-CinS & PraR [9]
The CinR-CinI-CinS system is regulated through quorum sensing, which leads to the production of the MetJ regulatory protein. The main function of CinR is to control quorum sensing by initiating the expression of the cinIS operon. Meanwhile, the primary role of CinI is to synthesize AHL, while CinS works by inactivating PraR, a repressor molecule, through the formation of a 1:1 dead-end complex.
Figure 6. Animation of PraR and CinS forming a 1:1 dead-end complex
AHL Synthesis [10 , 11]
The PraR repressor inhibits the activity of the praiI promoter, resulting in decreased production of MetJ as a consequence of reduced RaiI and RaiR levels. AHL synthesis is primarily carried out by RaiI, while RaiR functions as an AHL receptor that allows bacteria to respond to changes in population density. This response includes increased expression of CinI induced by AHL, resulting in higher levels of CinS expression, ultimately leading to increased MetJ expression. Furthermore, AHL can also be synthesized through the activity of the CinR-CinI-CinS system.
Figure 7. Animation of how AHL affects the relationship of CinR and CinI
CinS Expression Enhancement [12]
Even though CinI and CinS proteins can be produced independently, the production of CinS is still relatively low. Our team's objective is to avoid excess biofilm production, so we utilized the capability of the CinI protein to increase the production of CinS. This enhances the binding of CinS to PraR, thereby upregulating the raiR and raiR genes.
Figure 8. Animation of relationship between CinI and CinS
Biosafety Construct
Figure 1.c A diagram of our Biosafety construct
Our Biosafety construct is designed to localize in coral. The sequence of the construct was created by Johns Hopkins 2021 iGEM Team and then sent to IDT for synthesis. After receiving the synthesized construct, amplified and added complementary basepairs to pET28a and performed gibson assembly. This resulting plasmid was then introduced into E. coli DH5Alpha cells, followed by purification using miniprep technique and sequencing analysis to validate the correctness of the construct's sequence.
For cloning results, please visit the engineering page
The potential risks of GMOs in the natural environment and their impact on marine ecosystems have prompted our team to draw inspiration from the biosafety mechanism developed by iGEM John Hopkins 2021 Team [13], which utilizes quorum sensing. This mechanism enables us to regulate our bacteria based on population density. Antitoxins are constantly produced in the bacteria in small amounts. When the bacterial population reaches a high density, there is an increase in the concentration of autoinducers, specifically AHL molecules. This triggers the repression of toxins by our bacteria to allow the bacteria to survive; while the anti-toxins(MazE) will neutralize any remaining toxins(MazF). MazF functions as an mRNA interferase, and its activation under conditions of stress triggers a sequence-specific cleavage of mRNA molecules, resulting in a cessation of cellular growth. In the absence of stress, MazF is deactivated by forming a complex with its corresponding antitoxin, MazE.
Conversely, when the population density decreases due to factors like ocean currents causing bacteria dispersion from corals, minimal or no AHL molecules are received, toxins are being produced inside the bacteria at high amounts, overwhelming the amount of anti-toxins produced, leading to slow self-destruction overtime. This mechanism acts as a biosafety killswitch, effectively preventing unintended proliferation in other environments. Our project has undergone thorough consultation with renowned experts in the field, including Dr. Tang Sen-Lin and Professor David Bourne who have given their approval for its non-disruptive impact on the ecosystem. For more information, visit our Integrated Human Practice page.
Figure 9. Full animation of Biosafety construct
EsaI-EsaR-EsaBox [14]
The EsaI-EsaR-EsaBox system functions as a regulatory mechanism for the production of anti-toxins and toxins in bacteria. Previous studies have indicated that EsaR acts as a repressor by binding to the EsaBox region. Previous studies [15] have demonstrated that upon binding to EsaR in a 1:1 ratio, inducible molecules exhibit the ability to downregulate the production of our toxin, MazF. This is achieved through an upregulation in the synthesis of TetR protein. The key component involved in this process is the cognate transcriptional regulator encoded by EsaR, which responds to AHL signals. Additionally, EsaI encodes an AHL (N-acyl homoserine lactone) signal synthase responsible for synthesizing AHL molecules (EsaI).
Figure 10. Animation of EsaI-EsaR-EsaBox system
TetR & MazF Interaction
As mentioned previously, it has been observed that the TetR protein serves as a repressor to the tetR promoter (R0040). This interaction results in a decrease in MazF toxin production. The interaction between the TetR repressor and the MazF toxin is crucial for regulating toxin levels.
Figure 11. Animation of TetR & MazF interaction
MazE & MazF Interaction
As specified, MazF functions as the toxin that prevents any potential environmental harm caused by our engineered bacteria. MazE plays a crucial role as an antitoxin, maintaining a balanced and stable toxin-antitoxin system in high bacterial population density. The interaction between MazE and MazF is vital for maintaining a balanced toxin-antitoxin system and preventing potential environmental harm caused by the engineered bacteria. The MazE/MazF toxin-antitoxin system has been widely used in many different organisms [12]. Our biosafety system depends on regulation of the bacterial population.
Figure 12. Animation of MazE & MazF interaction
J23113 & TetR Promoter
We opted for the J23113 promoter as a suitable choice to control the production of MazE and EsaI proteins, primarily due to its characteristic as a "moderate-to-weak promoter". This selection ensures that the antitoxin is produced in limited quantities, effectively counteracting some of the MazF toxins. As a result, it prevents immediate bacterial death under any circumstances. In a similar fashion, we selected the tetR promoter(R0040) to regulate MazF and EsaR production based on its repressible nature and moderate to strong strength. This specific promoter's function can be repressed by TetR protein, providing an effective mechanism for regulating downstream gene expression.
For cloning results, please visit the engineering page
Functional Assays
The goal of our functional assays is to:
  1. Prove that our Biofilm construct produces biofilm.
  2. Prove that our MetJ construct can regulate biofilm growth rate.
  3. Prove that our Biosafety construct kills the bacteria beyond a certain concentration.
For functional assay results, please visit the proof of concept page
Biofilm Production Assay
We will culture E. coli DH5α containing the Csg construct and E. coli DH5α and compare their biofilm growth using an ELISA reader and crystal violet staining to ensure that the Csg construct does produce biofilm.
MetJ Protein Assay
Because we were not able to finish cloning our MetJ construct, we decided to transform the fragment that contains the metJ gene (MetJ fragment 3) into E. coli DH5α along with our Csg construct. This allows us to test whether expression MetJ proteins can successfully repress biofilm production. In this assay, we plan on growing multiple cultures of engineered E. coli containing MetJ fragment 3 and Csg construct, and a control group (only Csg construct), and measuring their bacteria population and amount of biofilm through ELISA reader with different staining methods. This allows us to see whether the MetJ regulatory protein can successfully repress biofilm gene expression. We expect to see a significant difference in biofilm growth in the cultures containing only the Csg construct and the cultures containing the metJ gene.
For functional assay results, please visit the proof of concept page
Biosafety Assay [14]
We want to demonstrate that our engineered E. coli bacteria die off at low culture concentrations and survive at high concentrations. To do this, we will measure the growth of E. coli containing our biosafety plasmid across various liquid culture concentrations. This not only allows us to perceive the effectiveness of our mechanism but also find whether the concentration in which the bacteria population starts to die out is too high or too low, which will be used to assist future optimizations of our project.
MetJ Assay
We aim to demonstrate that individual bacteria biofilm growth rates decrease at higher cell densities. To achieve this, we will cultivate engineered E. coli cultures (with MetJ and Biofilm plasmids) for varying durations. We will measure bacterial population and biofilm levels using an ELISA reader with different staining methods. This will enable us to track changes in bacterial population and biofilm production over time, helping us to calculate the individual bacteria biofilm production rate. We anticipate observing a decline in individual bacteria biofilm production rates as the bacterial population increases.
For functional assay results, please visit the proof of concept page
Implementation Testing
After we prove that our constructs work through our functional assays, we still need further testing before implementing our mechanism in real life. To ensure that our mechanism functions as intended in the ocean, we will re-run our functional assays in ocean water conditions. To ensure that our mixture of coral feed spray attaches to the mucus layer of the coral and that our probiotics are properly absorbed by the corals, we will test our mechanism on corals under a controlled setting.
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