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.
Cloning
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
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
Goal
The goal of our functional assays is to:
- Prove that our Biofilm construct produces biofilm.
- Prove that our MetJ construct can regulate biofilm growth rate.
- 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.
References
[1] Barnhart, M. M., & Chapman, M. R. (2006). Curli biogenesis and function. Annual review of microbiology, 60, 131–147.
https://doi.org/10.1146/annurev.micro.60.080805.142106
[2] Buchholz, H. H., Bolaños, L. M., Bell, A. G., Michelsen, M. L., Allen, M. J., & Temperton, B. (2022). A Novel and Ubiquitous Marine Methylophage Provides Insights into Viral-Host Coevolution and Possible Host-Range Expansion in Streamlined Marine Heterotrophic Bacteria. Applied and environmental microbiology, 88(7), e0025522. https://doi.org/10.1128/aem.00255-22
[3] Podar, M., May, A. L., Bai, W., Peyton, K., Klingeman, D. M., Swift, C. M., Linson, D. A., Mathieu, J., Siljeström, D., Beneyto, I., Stadler, L. B., Pinhas, Y., Löffler, F. E., Alvarez, P. J. J., & Kumar, M. (2021). Microbial diversity analysis of two full-scale seawater desalination treatment trains provides insights into detrimental biofilm formation. Journal of Membrane Science Letters, 1(1), 100001. https://doi.org/10.1016/j.memlet.2021.100001
[4] Brombacher, E., Baratto, A., Dorel, C., & Landini, P. (2006, March). Gene expression regulation by the curli activator CsgD protein: Modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion. Journal of bacteriology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1428138/
[5] Author links open overlay panelLimin Feng a b, a, b, c, d, years, A. recent, Alimba, C. G., Auta, H. S., Neto, J. A. B., Barboza, L. G. A., Capolupo, M., Curren, E., Dussud, C., Frère, L., Jin, Y., Kanki, M., Kirstein, I. V., Martínez-Gómez, C., Miao, L., … Ha, J. (2020, March 19). Investigating the composition and distribution of microplastics surface biofilms in coral areas. Chemosphere. https://www.sciencedirect.com/science/article/abs/pii/S004565352030758X
[6] Evans, J. S., Paul, V. J., & Kellogg, C. A. (2022, October 17). Biofilms as potential reservoirs of stony coral tissue loss disease. Frontiers. https://www.frontiersin.org/articles/10.3389/fmars.2022.1009407/full
[7] US Department of Commerce, N. O. and A. A. (2013, June 1). Zooxanthellae...what’s that - corals: NOAA’s National Ocean Service Education. Zooxanthellae: Corals Tutorial. https://oceanservice.noaa.gov/education/tutorial_corals/coral02_zooxanthellae.html
[8] The involvement of cell-to-cell signals in the development of ... - science. (n.d.-b). https://www.science.org/doi/10.1126/science.280.5361.295
[9] Frederix, M., Edwards, A., Swiderska, A., Stanger, A., Karunakaran, R., Williams, A., Abbruscato, P., Sanchez-Contreras, M., Poole, P. S., & Downie, J. A. (2014, July 2). Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins. PubMed Central (PMC). https://doi.org/10.1111/mmi.12670
[10] Edwards, A., Frederix, M., Wisniewski-Dyé, F., Jones, J., Zorreguieta, A., & Downie, J. A. (2009, March 6). The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI. PubMed Central (PMC). https://doi.org/10.1128/JB.01650-08
[11] Frederix, M., Edwards, A., McAnulla, C., & Downie, J. A. (n.d.). Co-ordination of quorum-sensing regulation in Rhizobium leguminosarum by induction of an anti-repressor. (PDF) Co- ordination of Quorum-sensing Regulation in Rhizobium Leguminosarum by Induction of an Anti-repressor | Marijke Frederix - Academia.edu. https://www.academia.edu/25167010/Co_ordination_of_quorum_sensing_regulation_in_Rhizobium_
leguminosarum_by_induction_of_an_anti_repressor
[12] Boss, L., & Kędzierska, B. (n.d.). Bacterial Toxin-Antitoxin Systems' Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology. MDPI. https://www.mdpi.com/2072-6651/15/6/380
[13] Team:hopkins. (n.d.). https://2021.igem.org/Team:Hopkins
[14] Minogue, T. D., Wehland, M., Bernhard, F., & Susanne. (2002). The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. Molecular Microbiology, 44(6), 1625–1635. https://doi.org/10.1046/j.1365-2958.2002.02987.x
[15] Susanne, Majerczak, D. R., & Coplin, D. L. (1998). A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proceedings of the National Academy of Sciences of the United States of America, 95(13), 7687–7692. https://doi.org/10.1073/pnas.95.13.7687
[2] Buchholz, H. H., Bolaños, L. M., Bell, A. G., Michelsen, M. L., Allen, M. J., & Temperton, B. (2022). A Novel and Ubiquitous Marine Methylophage Provides Insights into Viral-Host Coevolution and Possible Host-Range Expansion in Streamlined Marine Heterotrophic Bacteria. Applied and environmental microbiology, 88(7), e0025522. https://doi.org/10.1128/aem.00255-22
[3] Podar, M., May, A. L., Bai, W., Peyton, K., Klingeman, D. M., Swift, C. M., Linson, D. A., Mathieu, J., Siljeström, D., Beneyto, I., Stadler, L. B., Pinhas, Y., Löffler, F. E., Alvarez, P. J. J., & Kumar, M. (2021). Microbial diversity analysis of two full-scale seawater desalination treatment trains provides insights into detrimental biofilm formation. Journal of Membrane Science Letters, 1(1), 100001. https://doi.org/10.1016/j.memlet.2021.100001
[4] Brombacher, E., Baratto, A., Dorel, C., & Landini, P. (2006, March). Gene expression regulation by the curli activator CsgD protein: Modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion. Journal of bacteriology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1428138/
[5] Author links open overlay panelLimin Feng a b, a, b, c, d, years, A. recent, Alimba, C. G., Auta, H. S., Neto, J. A. B., Barboza, L. G. A., Capolupo, M., Curren, E., Dussud, C., Frère, L., Jin, Y., Kanki, M., Kirstein, I. V., Martínez-Gómez, C., Miao, L., … Ha, J. (2020, March 19). Investigating the composition and distribution of microplastics surface biofilms in coral areas. Chemosphere. https://www.sciencedirect.com/science/article/abs/pii/S004565352030758X
[6] Evans, J. S., Paul, V. J., & Kellogg, C. A. (2022, October 17). Biofilms as potential reservoirs of stony coral tissue loss disease. Frontiers. https://www.frontiersin.org/articles/10.3389/fmars.2022.1009407/full
[7] US Department of Commerce, N. O. and A. A. (2013, June 1). Zooxanthellae...what’s that - corals: NOAA’s National Ocean Service Education. Zooxanthellae: Corals Tutorial. https://oceanservice.noaa.gov/education/tutorial_corals/coral02_zooxanthellae.html
[8] The involvement of cell-to-cell signals in the development of ... - science. (n.d.-b). https://www.science.org/doi/10.1126/science.280.5361.295
[9] Frederix, M., Edwards, A., Swiderska, A., Stanger, A., Karunakaran, R., Williams, A., Abbruscato, P., Sanchez-Contreras, M., Poole, P. S., & Downie, J. A. (2014, July 2). Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins. PubMed Central (PMC). https://doi.org/10.1111/mmi.12670
[10] Edwards, A., Frederix, M., Wisniewski-Dyé, F., Jones, J., Zorreguieta, A., & Downie, J. A. (2009, March 6). The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI. PubMed Central (PMC). https://doi.org/10.1128/JB.01650-08
[11] Frederix, M., Edwards, A., McAnulla, C., & Downie, J. A. (n.d.). Co-ordination of quorum-sensing regulation in Rhizobium leguminosarum by induction of an anti-repressor. (PDF) Co- ordination of Quorum-sensing Regulation in Rhizobium Leguminosarum by Induction of an Anti-repressor | Marijke Frederix - Academia.edu. https://www.academia.edu/25167010/Co_ordination_of_quorum_sensing_regulation_in_Rhizobium_
leguminosarum_by_induction_of_an_anti_repressor
[12] Boss, L., & Kędzierska, B. (n.d.). Bacterial Toxin-Antitoxin Systems' Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology. MDPI. https://www.mdpi.com/2072-6651/15/6/380
[13] Team:hopkins. (n.d.). https://2021.igem.org/Team:Hopkins
[14] Minogue, T. D., Wehland, M., Bernhard, F., & Susanne. (2002). The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. Molecular Microbiology, 44(6), 1625–1635. https://doi.org/10.1046/j.1365-2958.2002.02987.x
[15] Susanne, Majerczak, D. R., & Coplin, D. L. (1998). A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proceedings of the National Academy of Sciences of the United States of America, 95(13), 7687–7692. https://doi.org/10.1073/pnas.95.13.7687