Team:FCB-UANL/Description
INSPIRATION
While we were looking for biological components capable of forming a foam, we found a set of proteins called Ranaspumins which are present in Leptodactylidae frog’s bubble nests. Their overall foaming and lectin (carbohydrate-binding proteins1 properties make them essential molecules for foam’s stability, due to their role building a scaffold for bubbles to form. In the following video of National Geographic, it can be seen the process by which the frogs form their foam nests.
However, we knew we had to look for a way to improve stability; so we considered B. subtilis’ natural metabolites, surfactin and biofilm, for this work. Considerations used for components’ selection are further explained in our Engineering success section.
BACKGROUND
The major regulation pathway involved is B. subtilis’ Quorum Sensing (QS) system, which regulates gene expression in response to cell density2. Its importance relies on cell differentiation, because surfactin and biofilm production are both related to QS. Due to the complexity of this regulation, we wrote the manual How to B. subtilis II, which includes all the concerns that have to be taken into account when working with B. subtilis’ natural networks. This manual was made as a result of all the processes documented in our Engineering success section, and it is available in the Contributions section.
COMPONENTS
These proteins are found in frogs’ foam nests. The female is in charge of disposing the eggs along with the foam precursors, while the male frog releases its sperm and whips the foam precursors with its legs to generate a white foam1,3. It is worth mentioning that the foam is very stable in the natural environment, since it can last approximately 10 days in nature3. The foams’ main components consist of carbohydrates and six proteins: Ranaspumins 1-61.
Through several analyses, many roles were proposed for these six proteins. Rsn-1 presents a sequence similarity to cysteinyl proteinases. Rsn-2 has been proved to have surfactant properties, thus being the protein that has been besought to be the main molecule causing the foam to form. Finally, Rsn-3 to Rsn-6 have been found to have similar structures to those of lectins, therefore suggesting a role as foam stabilizers or even defense against pathogens1. To explain Ranaspumin-2’s longer durability in the environment, it has been suggested that it interacts with the lectin-like proteins, which bind to the polysaccharides present in the foam, conferring stability to it, as we can see in the image1.
We will use the well known chassis E. coli to produce the selected ranaspumins. For both strains, the expression of the proteins will be controlled by a well characterized vanillate inducible promoter, pVanCC, which has a high dynamic range to control the production4. This promoter is regulated by VanRAM. On the other hand, the RBS’s of the ranaspumins were designed with the RBS calculator tool developed by Sallis5. Proteins also carry a 6xhis tag with a thrombin cleavage site for further purification with nickel columns. The terminator used is rrnb T1(BBa_B0010).
The promoter and RBS used for the regulator VanRAM are PlacIQ (strong, constitutive) and van2, respectively. These are the same used by Meyer and collaborators4 to characterize the strength and dynamic range of pVANcc.
In particular, Rsn-2 (the main foamy agent) will be produced individually on a plasmid with low number of copies (pSB3K3).
Similarly, Rsn-3, Rsn-4 and Rsn-5 (foam stabilizers) will be produced together on a plasmid with low number of copies (pSB3K3) in a separate strain.
Another molecule that caught our attention is surfactin. It is one of the most effective biosurfactant molecules, since it is able to reduce surface tension for as much as 27mN/m, and it has a high emulsifying activity6,7, therefore promoting foam formation. Because of its properties, surfactin seems to be a good complement for Rsn-2 as a secondary foaming agent in our project.
Naturally occurring surfactin production is a process deeply connected with quorum sensing, and it all starts when the bacteria density in the medium increases, causing more comX pheromone to be found in it. Then, this molecule causes the phosphorylation of comP, which further phosphorylates comA. The phosphorylated form of comA initiates the transcription of the surfactin operon8.
For us to control this network, we plan to take advantage of two groups of molecules closely connected: Rap proteins and Phr peptides. Rap (aspartyl-phosphate phosphatases) proteins dephosphorylate molecules such as spo0A~P and comA~P. The counterpart, the Phr peptides, consists of peptides matured extracellularly which inhibit the Rap proteins’ function.
With this in mind, we will overexpress PhrC (also known as CSF), whose function is to inhibit the RapC protein, which is a negative regulator of ComA-P, thus allowing comA~P to continue with the transcription of the srf operon9.
Besides increasing the expression of the surfactin operon, we will overexpress two other genes: Sfp and AbrB. The Sfp gene product activates the surfactin synthase enzyme (which is produced by the srf operon), and AbrB is a master regulator of the transcription of genes expressed during the transition state between growth and the beginning of sporulation. Previously, Team Lyon-INSA 2012 demonstrated that these pieces together enhance surfactin production.
The following diagram summarizes how we established a plan for enhancing surfactin production.
The next figure represents the sequence that will be integrated for Surfactin enhancement. Proteins PhrC (quorum sensing regulation) and Sfp (surfactin synthetase activator) will be regulated through a xylose inducible promoter, which is regulated by XylR. On the other hand, AbrB (biofilm inhibitor) will be controlled by the constitutive promoter P43, which is active during exponential and lag phases. Spo0E is a delayer of sporulation, AmyE-5’ and AmyE-3’ are the homologies for genome integration and cat is a chloramphenicol resistance. All the RBS’s except in the case of XylR and cat were designed with the RBS calculator.
Due to the lectin-like properties of the ranaspumins, the presence of polysaccharides in the mixture is essential for the foam’s stability. Going back to B subtilis, we learned there is a subset of cells within the colony that secrete extracellular biofilm’s compounds, mainly polysaccharides and proteins, which confer protection and stability10. Two operons produce these extracellular compounds: the eps and the tapA operons11.
This network starts with several external signals which cause the phosphorylation of several kinases (KinA, KinB, KinC, KinD, and KinE). They further start a phosphorelay and produce spo0A~P through intermediaries spo0F and spo0B11,12. When spo0A~P is found in medium levels, the bacteria are dedicated to biofilm development; once the levels of spo0A~P are sufficiently high, they commit to sporulation13, which is not desired due to safety measures.
Throughout this process, pairs of transcription factors are playing a key role in controlling the entry into sporulation, and they are sinI-sinR and abbA-abrB. They both act similarly: sinR and abrB are repressor proteins which hinder biofilm development and entry into sporulation, while sinI and abbA are their respective counterparts11,14.
Genetic regulation of biofilm formation by the organism B. subtilis; This production is carried out by two operons eps and TapA.
Taking this into consideration, we saw an opportunity to use sinI and abbA; we considered that the expression of these two proteins could enhance biofilm production. Hence, the following diagram represents the circuit we designed to help us achieve the previously mentioned objective.
The following figure is a representation of the sequence that will be integrated for Biofilm enhancement. Proteins AbbA (biofilm regulator) and SinI (biofilm regulator) will be regulated through a xylose inducible promoter, which is regulated by XylR. In this piece AbbA and SinI proteins will be produced, which inhibit the pieces SinR and AbrB, genes recognized by the inhibition of biofilm production. Spo0E is a delayer of sporulation, AmyE-5’ and AmyE-3’ are the homologies for genome integration and cat is a chloramphenicol resistance. All the RBS’s except in the case of XylR and cat were designed with the RBS calculator.
SPORULATION DELAY
Sporulation is a phenomenon which we want to avoid as part of the safety measures and to orientate the bacteria’s efforts towards producing the desired components. Spo0A~P is the master regulator in charge of coordinating entry into sporulation. To counteract this property, we plan to express spo0E in both of B. subtilis’ strains under the control of a low level-spo0A~P inducible promoter. This protein dephosphorylates spo0A~P, master regulator for entry into sporulation, so our system will delay sporulation by dephosphorylating spo0A~P when its concentration starts to increase13.
REFERENCES
1. Fleming, R. I., Mackenzie, C. D., Cooper, A., & Kennedy, M. W. (2009). Foam nest components of the tungara frog: a cocktail of proteins conferring physical and biological resilience. Proceedings of the Royal Society B: Biological Sciences, 276(1663), 1787-1795.
2. Lazazzera, B. A. (2000). Quorum sensing and starvation: signals for entry into stationary phase. Current opinion in microbiology, 3(2), 177-182.
3. Cooper, A., Vance, S. J., Smith, B. O., & Kennedy, M. W. (2017). Frog foams and natural protein surfactants. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 534, 120-129.
4. Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196-204.
5. Salis, H. M. (2011). The Ribosome Binding Site Calculator. Methods in Enzymology Synthetic Biology, Part B - Computer Aided Design and DNA Assembly, 19-42. doi:10.1016/b978-0-12-385120-8.00002-4
6. Gudiña, E. J., Fernandes, E. C., Rodrigues, A. I., Teixeira, J. A., & Rodrigues, L. R. (2015). Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium. Frontiers in Microbiology, 6, 59.
7. Shaligram, N. S., & Singhal, R. S. (2010). Surfactin–a review on biosynthesis, fermentation, purification and applications. Food technology and biotechnology, 48(2), 119-134.
8. Wu, Q., Zhi, Y., & Xu, Y. (2019). Systematically engineering the biosynthesis of a green biosurfactant surfactin by Bacillus subtilis 168. Metabolic engineering, 52, 87-97.
9. Pottathil, M., & Lazazzera, B. A. (2003). The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis. Front Biosci, 8, d32-d45.
10. Branda, S. S., Vik, Å., Friedman, L., & Kolter, R. (2005). Biofilms: the matrix revisited. Trends in microbiology, 13(1), 20-26.
11. Cairns, L. S., Hobley, L., & Stanley‐Wall, N. R. (2014). Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms. Molecular microbiology, 93(4), 587-598.
Burbulys, D., Trach, K. A., & Hoch, J. A. (1991). Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell, 64(3), 545-552.
13. Fujita, M., González-Pastor, J. E., & Losick, R. (2005). High-and low-threshold genes in the Spo0A regulon of Bacillus subtilis. Journal of bacteriology, 187(4), 1357-1368.
14. Tucker, A. T., Bobay, B. G., Banse, A. V., Olson, A. L., Soderblom, E. J., Moseley, M. A., ... & Cavanagh, J. (2014). A DNA mimic: The structure and mechanism of action for the anti-repressor protein AbbA. Journal of molecular biology, 426(9), 1911-1924.