how to bacillus subtilis ii

How to Bacillus subtilis II Team iGEM UANL-FCB 2020

This manual is intended to provide an easy introduction to work with Bacillus subtilis

and its genetic network to future iGEM teams. iGEM team 2016 Bonn and Freiburg

previously developed a manual which addresses some topics related to the

experimental work with Bacillus subtilis , including some useful tips and protocols. We

tried to complement this previous work creating How to Bacillus subtilis II. Here, we

will address some aspects of the genetic network that controls the fate of Bacillus

subtilis cells, a guide on how to perform genome integration in this bacteria, and a brief

resume of useful tools you should have into consideration. We will also include a

systematic review of previous iGEM projects developed on Bacillus subtilis .

We hope this manual will be useful for future igemers, in particular if you have never

worked before with B. subtilis .

Team iGEM FCB-UANL 2020

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Index

Bacillus subtilis overview 4

Characteristics regarding the species 4 Characteristics regarding the genus 4 Bacillus Subtilis’s ID 5

Quorum sensing and population subspecialization 6

The start of the genetic network: Quorum sensing 6 System ComQXPA in detail 7

Subpopulation types 8

Motile bacillus 8 Understanding motility and mobility in bacteria 8

What does bacterial motility serve for? 8 Which Bacillus are motile? 9 Genes involved in B. Subtilis motility 9

Surfactin producer cells 10 Regulating Surfactin 10 Bacillus subtilis as an antibiotic producer 11

Biofilm producer cells 11 Cannibal cells 12 Exoprotease producer cells 13 Sporulating cells 14

B. subtilis plasmids 16

Replicative plasmids 16 Integrative plasmids 16

Useful tools for Bacillus subtilis 18

Genetic elements for Bacillus subtilis 18 Names 18 Bacillus SEVA siblings 20 Subtiwiki 20 BsubCyc 22 Bacillus Genetic Stock Center 22

Previous iGEM Bacillus subtilis Projects 24

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2016 24 2017 25 2018 27 2019 29

References 31

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Bacillus subtilis overview Characteristics regarding the species

Bacillus subtilis ( B. Subtilis) (Fig. 1) is one of the most common bacteria in the world, often referred to as ubiquitous, although it is found predominantly on soils. This organism, described by German scientist F. Cohn in 1872, consists of a rod-shaped and spore-forming Gram-positive bacterium. Since the discovery of the strain 168 in 1958 by Spizizen, an immense amount of research regarding this organism has been conducted over half a century. B. Subtilis serves as a model for study of spore formation and low GC% Gram-positive bacteria (Piggot, P., 2009).

Figure 1. Electron microscopy images of B. subtilis. Taken by Thierry meyiheuc. The image is reproduced from Chastanet and Carballido-Lopez (2012).

This organism has the capacity to grow in nutrient media and in chemically defined salt media as well. On chemical-based media, simple sugars like glucose and malate provide sources of carbon and ammonium salts or certain amino acids as sources of nitrogen. B. subtilis strain 168 , on which most studies are performed, is a tryptophan auxotroph (trpC2) and therefore requires the addition of tryptophan to the growth media, even those containing acid-hydrolyzed proteins such as casein (Harwood, C. 2013).

Characteristics regarding the genus

The genus Bacillus has around 318 described species due to recent taxonomic changes, which attracted attention from the public because of its economic and medical importance (Elshaghabee, F. 2017). Efforts have been made to differentiate pathogenic and probiotic species, some of them include phylogenetic discrimination, which has allowed the proper identification of strains for further usage.

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The pathogenic characteristics depends over strain and variety specific production of several extracellular factors (phospholipase, cereulide, enterotoxin Hbl, non-haemolytic toxin [Nhe], etc.) having role in cellular membrane disruption and induction of necrotic enterocolitis cytotoxin (Elshaghabee, F. 2017). Probiotic species, as many studies indicate, has an increased number of health benefits including immune modulation on Lactobacillus and lowering of plasma triglycerides.

Bacillus Subtilis ’s ID

As previously stated, B. Subtilis is very easy to find on soils and in laboratories, thus its taxonomic identification has been studied for many years now, Table 1 provides a summary of B. Subtilis taxonomic information. The genome sequence of B. subtilis 168 was completed in June 1997. This strain is a tryptophan auxotrophic mutant derived from the original B. subtilis ATCC 6051 which was isolated from boiled hay infusion by Cohn in 1872 (Wipat, A. 1998).

Scientific Classification

Domain Bacteria

Phylum Firmicutes

Class Bacilli

Order Bacillales

Family Bacillaceae

Genus Bacillus

Species B. Subtilis

Table 1. Taxonomic information of B. subtilis. Information extracted from Bergey’s manual of systematic bacteriology: volume 3: The Firmicutes.

One of the most important aspects of B. Subtilis ’s taxonomy is being part of the phylum of Firmicutes , which differentiates from E. Coli , which is part of the phylum of

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Proteobacteria . The discriminatory factor between these phyla is the spore formation , Firmicutes can form endospores and are mostly Gram-positive, whereas Proteobacteria species do not form spores and are completely Gram-negative.

Firmicutes’ spores contain enough energy for germination and can adapt to quickly respond to substrate availability and formation of a vegetative cell able to replicate. Therefore, B. Subtilis and other firmicutes can easily outgrow other organisms after transfer to microbiological media, explaining their ubiquitous characteristic (Parkes, R. 2009).

Quorum sensing and population subspecialization Bacillus subtilis cells can differentiate into different kinds of cellular types. Cellular fate depends on the levels and phosphorylation of three main master regulators: ComA, Spo0F y DegU . Surfactin producers and competent cells depend on ComA, biofilm producers, cannibal cells and sporulating cells depend mainly on Spo0A and exoprotease producer cells depend on DegU . Each type of subpopulation and regulation system will be discussed below.

The start of the genetic network: Quorum sensing

The ability to alter gene expression and behaviour, depending on the environment, is characteristic of bacteria. One of the most dynamic changes they face is population density. Bacteria can exist in small populations, however, as their population density increases, they can coexist with other bacterial species. In these interactions, bacteria sense and respond to outside changes, using a cell-cell communication that is known as quorum sensing (QS). This communication pathway produces, detects, releases and responds with molecules known as "auto-inducers". Communication through quorum sensing allows bacteria to coordinate the expression of genes from an entire community. The first quorum-sensing circuit was identified in Vibrio fischeri in 1983 (Engebrecht et al ., 1983). Nowadays, dozens of bacterial quorum-sensing circuits have been identified, and all of them allow bacteria to accomplish the same task; to regulate gene expression according to the population density with which they coexist (Bassler et al., 2013).

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Bacillus subtilis codes for two types of quorum-sensing, ComQXPA and Rap-Phr, both of which control a regulator called ComA, which is responsible for surfactant production. Genetically, the ComQXPA system is coded for the operon comQXPA. The Rap-Phr system, on the other hand, codes for Rap receptors and Phr auto-inducers (Bareia et al., 2017).

System ComQXPA in detail

There are four main genes involved in the Quorum Sensing System: ComQXPA ; with one common promoter in front of ComQ and two putative ones in front of ComX and ComA. Even though there are several polymorphisms between strains, four phenotype groups have been identified, and communication is possible between strains of the same group (Dogsa, et al. 2014).

ComX is the main system component: it is a pheromone that works as an extracellular signaling molecule. Among the responses comX activates, surfactin production is notable, as well as biofilm production (indirectly) and extracellular DNA release. It also has an important role in cell differentiation . The sequence presents marked polymorphism, but in all strains a tryptophan residue is present, which is modified by ComQ (Okada, et al. 2005)

ComQ encodes an enzyme: isoprenyl transferase that is in charge of comX exportation and postranslational modification of the produced precursor; these is the action to attach an isoprenyl unit to the tryptophan residue (the specific mechanism can vary from one strain to the other) (Dogsa, et al. 2014). Its activity is still on review, since the specific mechanism of comX exportation is still unknown (Okada, et al. 2005) in addition to the fact that some databases indicate that it may have a membrane-binding region (Dogsa, et al. 2014).

ComP is a membrane receptor that recognizes comX and then activates comA through phosphorylation. It has a histidine-kinase and an ATP-binding domain (Spacapan, et al. 2018).

ComA represents the final step of the QS pathway: it is a transcriptional response regulator that activates approximately 89 genes in 35 operons. Some of these are srfA (surfactin production), degQ (exoproteases production) (Spacapan, et al . 2018).

The main regulation consists of a negative feedback loop based on ComX production of the productive cell (Dogsa, et al . 2014)

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Subpopulation types

Motile bacillus

This type of cellular subpopulation does not depend on the three master regulators mentioned above, which are present mainly on post-exponential growth phases. Instead, motility is developed during the exponential growth, and it has been suggested that these cells will later differentiate into the other subpopulations (Verhamme et al ., 2007; Vlamakis et al., 2008). Lets first review some basic concepts about bacterial motility.

Understanding motility and mobility in bacteria

Motility is the ability of living systems to exhibit motion and to perform mechanical work at the expense of metabolic energy. (Day, R., n.d.). Be sure not to confuse “motility” with “mobility”, because they´re not the same. The difference between both can be observed when analyzing the bacterial motion under a microscope. Brownian motion of particles demonstrates their mobility under the influence of thermal agitation . Likewise, motility, coming from the etymological root mot-, “to push or move”, meaning like this, “ability of automatic move”, (Online Etymology Dictionary, 2020) refers to the saltatory motion of the bacteria, it may transport the same particles to much greater distances using metabolic energy . (Day, R., n.d.). In summary, the mobility refers to the capacity of an organism to be moved, and motility to the ability of moving by itself.

What does bacterial motility serve for?

The usage of the motility in bacteria, mainly consists of conferring a bacteria to change direction . The importance of this resides in the fact that bacteria may require moving away or closer towards repellents or attractants, respectively. Like this, motile bacteria are effective root colonizers and can swim towards root exudates or other nutrient gradients earlier than non motile bacteria. (Bhawsar, S., 2011)

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Which Bacillus are motile?

All the members of the genus Bacillus have that characteristic rod-like shape. However, B. subtilis is one of the species of this genus that has been packed additionally with several flagella (whip-like tails). B. subtilis has peritrichous flagella, meaning the cell is covered with tiny tails. These tails can be seen using a light microscope with a specialized strain. (Steele, E., 2016). As another example, Bacillus piliformis, another bacteria from the Bacillus genus, possesses peritrichous flagella, and is therefore also motile. (DeLong, D. & Manning, P, 1994).

Genes involved in B. Subtilis motility

Among populations of B. subtilis , only a fraction of cells express sigD , the sigma factor necessary for flagellar production, resulting in heterogeneity in motility (Kearns, 2005). Some of these cell types can be distinguished from their sister cells because their altered gene expression results in morphological changes that are visible under the microscope. (Lopez, D., et al., 2009) Motility requires the induction of a large fla-che operon , which contains 31 genes encoding for proteins that make up the basal body of the flagella, the chemotaxis system , and the sigma factor SigD (Lopez, D., et al., 2009) SigD is encoded at the end of the fla-che operon and is required for the expression of the hag locus , which encodes flagellin, the protein comprising the actual flagellar filament, as well as for the motA and motB genes, which encode for the motor proteins necessary for flagellar rotation (Marquez-Magana & Chamberlin, 1994). Transcription of genes important for motility and matrix production is regulated by Spo0A . High levels of Spo0A-P repress the fla/che motility operon , whereas Spo0A-P is required for extracellular matrix gene expression via the activation of the regulatory protein SinI. (Lopez, D., et al ., 2009)

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Surfactin producer cells

Surfactin is an antibiotic and biosurfactant lipopeptide produced by B. subtilis, consisting of the anion of seven membrane peptides and a mixture of hydrophilic fatty acids (Stein, 2005).

Surfactin lipopeptide is the most powerful biosurfactant known, as a 20-M solution can decrease the surface tension of water from 72 mN m.1 (Carrillo et al ., 2003). Surfactin exerts an action like that of detergents in biological membranes and is distinguished by its exceptional emulsifying, foaming, antiviral capacity (Peypux et al., 1999).

Regulating Surfactin

The expression of srfA and comS is regulated by a complex network that handles cellular differentiation, including quorum sensing which operates from the extracellular concentration of ComX and the regulatory components of the ComXPA system mentioned before.

When the cell density is high, a large amount of extracellular ComX is concentrated so ComP begins to phosphorylate ComA which is the transcription factor of the operon srfA (responsible for producing surfactin). When the cell density is low (this is, during exponential phase) then ComP does not phosphorylates ComA and the surfactin production does not start.

Figure 2. Regulation of srfA operon. The schematic model for the regulation of the transcription of the srfA operon network involved in two extracellular signaling peptide-mediated quorum sensing in B. subtilis. Retrivered from Shoung Li, 2019.

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Bacillus subtilis as an antibiotic producer

Besides surfactin, it is worth to mention the potential of B. subtilis to produce antibiotics has been recognized for 50 years. Peptide antibiotics represent the predominant class as they have high rigidity, hydrophobicity, and/or cyclical structures with constituents such as D-amino; These structures are generally resistant to hydrolysis by peptidase or proteases (Katz and Demain, 1997). Cysteine residues can be oxidized by bisulfates or undergo modifications to a characteristic C-S intramolecular anchorage, obtaining resistance to oxidation.

B. subtilis uses two biosynthesis pathways for these antibiotics: 1) non-ribosome synthesis of peptides by mega enzymes (non-ribosomal peptide synthesizes) and 2) Ribosome synthesis of precursors, which are exposed to post-translational modifications and protein processing (Stein, 2005).

Figure 3. Antibiotic production regulatory routes. Regulatory routes of biosynthesis of B. subtilis antibiotics such as surfactin, subtilisin, surfactin, Skf death factor, and antimicrobial peptide related to Tas A spores. Arrows represent positive regulation and T-lines represent negative regulation. (Retrieved from Hamoen et al. (2003).

Biofilm producer cells

When bacteria are faced with hostile environments, they are prone to develop survival strategies, such as sporulation or the formation of extracellular matrix layers of proteins, exopolysaccharides and sometimes extracellular DNA, known as biofilm (Flemming et al. 2016). Bacteria in biofilm are generally more resistant to environmental

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stress and less susceptible to the effects of antibiotics (Costerton et al. 1999). At least five steps can be considered crucial for biofilm formation, (1) reversible attachment, (2) irreversible attachment, (3) microcolony formation, (4) biofilm maturation, and (5) dispersion (Stoodley et al. 2002). Particularly in Bacillus subtilis , transcription regulation is crucial for biofilm formation (Cairns et al . 2014). The transcriptional regulator Spo0A is critical in biofilm initiation. There are two repressors of biofilm formation: abrB and sinR, which act directly or indirectly on the 15 genes of the eps operon required for extracellular polysaccharide biosynthesis, within the tapA-sip-tasA operon, as well as the bslA gene encoding hydrophobic proteins. There is an antagonist that acts directly on the SinR biofilm repressor, known as SinI, which blocks the formation of SinR tetramers that interrupt the formation of biofilm, promoting biosynthesis and organization of the biofilm. In combination with eps, tasA is the major component of biofilms (Branda et al. 2001), tapA represents a component in lower concentration that anchors fibers in bacterial walls and forms tasA.

Cannibal cells

We already know that when forming a biofilm, a colony of Bacillus subtilis can differentiate into different subpopulations which are dedicated to perform different tasks (Kearns and Losick, 2005; Branda, et al., 2001; Shank and Kolter, 2011). Surprisingly, one of those subpopulations consist of bacteria which show cannibalistic behaviors (Mielich-Süss and Lopez, 2015). But, exactly on what does this form of cannibalism consist?

To understand this process better, we must first remember that this occurs in the onset of sporulation (Höfler, et al ., 2016). Sporulation in Bacillus subtilis is induced since it undergoes carbon, nitrogen, and phosphorus starvation (Piggot and Hilbert, 2004; Higgins and Dworkin, 2012). Taking this into consideration, cannibalism in sporulating bacteria, such as Bacillus subtilis, has been considered a countermeasure to delay sporulation as long as possible, since the latter is an energetically-expensive event (González, et al ., 2003; Höfler, et al ., 2016).

Now, the bacteria have a specialized mechanism that depends on both environmental factors and correct timings on the expression of genes. First, we need to have in mind that the operons related to cannibalism are skf and sdp . Basically, these operons

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consist of the toxins and the machinery needed to produce, release, and acquire resistance to them (González, et al., 2003; Ellermeier, et al., 2006).

But, when does the bacteria know how to express them both? It starts with the signaling molecule surfactin , which triggers matrix and cannibal toxin production in the same sub-population of cells by activation of KinC (López, et al., 2009), which further phosphorylates Spo0A, the master sporulation regulator, through a phosphorelay pathway (González, et al ., 2003; Piggot and Hillbert, 2004; Higgins and Dworkin, 2012). Phosphorylated spo0A (spo0A~P), when present in low levels, is in charge of activating transcription of the sdp and skf operons, either by indirect, -through AbrB inhibition- or direct positive regulation (Fujita, et al ., 2005). Therefore, cells that start phosphorylating spo0A lyse those who still have not started to phosphorylate the transcription factor (González, et al ., 2003). In Figure 4 you can see an oversimplified image based on the different authors cited before.

Figure 4. Simplified representation of the genetic regulation of cannibalism operons’ expressions. Adapted from: González, J. E., 2011; Lopez, D., 2009.

Exoprotease producer cells

Some of the important components of the biofilm are exoproteases, having two main enzymes: aprE (serine exoprotease or subtilisin) and NprE (metalloprotease). DegQ gene is responsible for protease production. aprE and NprE activity represents 95% of the total proteolytic activity; their importance lies in the role they play in supplying aminoacids for growth, but also in ComX degradation, thus regulating the Quorum Sensing System (Spacapan , et al. 2018).

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Exoprotease production occurs only during the post-exponential growth phase; and is regulated indirectly by ComX , as DegQ is regulated by comA regulon. Both AprE and NprE prevent autolysis of cells in the stationary phase and are not essential in growth nor sporulation, but some of their regulators are (Barbieri, et al. 2016).

Figure 5. Exoprotease regulation in B. subtilis. Retrieved from (Spacapan, et al. 2018) AprE is important for CSF and PhrA production, which are two QS signaling peptides; it is directly repressed by AbrB, ScoC and SinR, and activated by phosphorylated DegU . Among the indirect regulators, phosphorylated Spo0A, AbbA , phosphorylated SalA, TnrA, SinI, DegS, DeqQ, DegR and RapG are found (Barbieri, et al. 2016).

Sporulating cells

As mentioned before, sporulation is a stress response of many bacteria, including Bacillus subtilis , triggered by nutrient starvation (Piggot and Hilbert, 2004; Higgins and Dworkin, 2012). The process can take several hours, and the result is a spore which is resistant to many harsh environmental conditions. As sporulation is a very complex process, it is described in a broad perspective in this work. For further information, you may consult Piggot and Hilbert’s (2004a, 2004b), Setlow’s (2003), Nicholson and collaborators’ (2000), among others’ works.

At the beginning, a phosphorelay occurs when kinases (KinA, KinB, KinC, KinD, and KinE ) phosphorylate spo0F, which furthers phosphorylates spo0B which finally transfers the phosphate group to spo0A (Burbulys, et al ., 1991; Piggot and Hilbert, 2004a). When present in low levels, spo0A~P induces matrix production and cannibalism in a subpopulation of Bacillus subtilis ; when spo0A~P reaches a

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high-threshold , then the genes that are involved in the second stage of sporulation start to express (Fujita, et al ., 2005; López, et al ., 2009; Errington, 1993).

Once the second stage of sporulation begins, the bacteria start to develop a constant “communication” among the mother cell and the forespore. Differential expression of transcription factors along with other spo genes help in the development of the spore (Errington, 1993; Piggot and Hilbert, 2004a); a diagram depicting the previously mentioned process can be seen in Fig. 6. At the end, there is a spore resistant to very adverse environmental conditions within a mother cell that will eventually lyse (Piggot and Hilbert, 2004a; Errington, 1993) (Fig. 7).

Figure 6. Diagram that shows the “communication” among the prespore and the mother cell. This diagram was retrieved from: Piggot, P. (2004a)

Figure 7. Graphic representation of the different stages of sporulation and the genes related to each one. The diagram was retrieved from: Errington, J. (1993).

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B. subtilis plasmids

Replicative plasmids

Replicative plasmids for B. subtilis need a different origin of replication than plasmids for gram negative bacteria such as E. coli. There exist three main types of origin of replication for circular plasmids, theta, rolling circle and strand displacement. Most of the plasmids that have been characterized for gram positive bacterias have plasmids which use a mode of replication called rolling-circle (del Solar et al ., 1998). Given that some authors have reported problems with plasmid instability (Bron, et al., 1998), an usual strategy is to use shuttle vectors for E. coli and B. subtilis. This means that your replicative plasmid for B. subtilis generally should have an E. coli origin of replication for cloning and a origin of replication compatible with B. subtilis. iGEM team Toulouse 2016 created a part (BBa_K1937002) to turn any pSB1C3-based plasmid into a shuttle vector for E. coli and B. subtilis. This part includes the repU origin of replication and a kanamycin resistance gene for B. subtilis.

Integrative plasmids

This is the most commonly used method for modifying B. subtilis . These plasmids carry an origin of replication and resistance marker for E. coli , and sequences with homology to a part of the genome of B. subtilis. Generally this is done through double-crossover recombination. In this case, homology sequences bigger than 400 bp flank the genes that you desire to integrate, including a resistance marker. Your genes will be integrated between the respective homologies. In this type of integration it is important to linearize your plasmid (in a region that will not be integrated) to avoid single-crossover recombination (Harwood et al., 2013).

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Figure 8. B. subtilis double crossover recombination. Taken from Harwood et al., 2013. Some of the most common genes for integration into the B. subtilis genome are AmyE and ThrC . Integration in AmyE is easily detected through the amylase test and the 5’ and 3’ sequences are available in the part registry with the numbers BBa_K143001 and Ba_K 143002. ThrC creates threonine auxotrophic mutants, which can be detected by growing the strains in medium without threonine and medium with it.

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Useful tools for Bacillus subtilis B. subtilis is a model organism for gram positive bacteria, and it also has been widely commercially exploited. As a consequence, multiple tools have been developed to facilitate the work of this organism. Here we will list the ones that we found most useful.

Genetic elements for Bacillus subtilis

First of all, we recommend you to search in the official section for Bacillus subtilis parts in the parts registry: http://parts.igem.org/Bacillus_subtilis. Besides the parts registry, several authors intended to find and characterize genetic elements for B. subtilis . These elements are described in the following table.

Names Features and limitations Applications Refs

Native promoter library-1

Features: (1) 84 promoters with 3 orders of magnitude in the variation of maximal expression strength. Limitations: (1) Lack of promoters for dynamic regulation of gene expression. (2) Long sequences of promoters causing difficulties in regard to genetic manipulation.

(1) Enhancing heterologous protein expression. (2) Static regulation of gene expression level for metabolic engineering. (3) Genetic circuit construction.

Song et al. (2016)

Native promoter library-2

Features: (1) 114 promoters classified into 4 categories based on their active phase from exponential phase to stationary phase. Limitations: (1) Long sequences of promoters (300 bp) that are inconvenient for use in genetic manipulation.


Yang et al. (2017)

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http://parts.igem.org/Bacillus_subtilis

Synthetic promoter library-1

Features: (1) 32 synthetic promoters with 900-fold differences strength, which consisted of short promoter sequences (~60bp) for convenient genetic manipulation. Limitations: (2) Lack of promoters for dynamic regulation of gene expression.


Guiziou, et al. (2016)

Synthetic promoter library-2

Features: (1) 220 synthetic promoters with 140-fold differences strength, which consisted of short sequences (54-220 bp) for convenient genetic manipulation. Limitations: (1) Lack of promoters for dynamic regulation of gene expression

(1) Enhancing heterologous protein expression (2) Static regulation of gene expression level for metabolic engineering. (3) Genetic circuit construction.

Liu, et al. (2018)

Synthetic expression modules from up element to spacer sequence between RBS and the first codons

Features: (1) 12 000 synthetic expression modules with 5 orders of magnitude in variation of expression strength, inducing 32 synthetic expression modules for significant enhanced expression. Limitations: (1) Lack of modules for dynamic regulation of gene expression.


Sauer, et al. (2018)

RBS sequence library

Features: (1) 31 synthetic RBS sequences with 800-fold strength differences. Limitations: (1) Effects of 5’ end of the coding sequences on translation were not considered.



Synthetic proteolysis tag library

Features: (1) 22 synthetic proteolysis tags with 100-fold strength differences. Limitations: (1) Lack of proteolysis tags for dynamic regulation of gene expression.



Table 2. Developed genetic elements for regulation in Bacillus subtilis. Taken from Liu et al., (2018).

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Bacillus SEVA siblings

Bacillus SEVA siblings is a toolbox designed by Radeck et al. (2017) for the creation of integrative vectors for Bacillus subtilis based on the Standard European Vector Architecture (SEVA). It is designed so each integrative plasmid consists of four parts: a part containing a resistance gene and origin of replication for E. coli , a part with the 5’ region of a gene of your choose for integration, a part with the 3’ region and a part with a resistance gene for Bacillus subtilis and a multiple cloning site. This allows you to create integrating vectors targeted to any part of the genome that you desire. The assembly of these four parts is carried out with a golden gate reaction, which makes this toolkit very practical. The vectors created by the authors are available in the BGSC and the SEVA collection.

Figure 9. Parts of the Bacillus SEVA siblings vectors. E. coli origin of replication and resistance gene (blue), 5’ homology for integration (yellow), B. subtilis resistance gene and multiple cloning site (orange), 3’ homology for integration (red). Taken from Radeck et al., 2017.

Subtiwiki

Subtiwiki is a gene and protein-centered database for B. subtilis ( Flórez , et al., 2009) ). You can search for a specific protein and it will give you basic information such as locus, isoelectric point, molecular weight, function and links to externals databases and also publications related to that protein. This is a very complete database. It also includes a pathway browser, interaction browser, expresion browser, genome browser, regulation browser and a list of the genes in the regulon of your protein.

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The regulation browser gives you a quick perspective on the interactions of your gene, it represents activation with green lines and represion with red lines. As a plus, you can export this data as a CSV file.

Figure 10. Regulation network of Spo0A. Each blue dot is a protein, activation is represented with green lines and repression with red lines. Retrieved from: Subtiwiki (Zhu B & Stülke, 2018). In the expression browser section you will find data on the transcription and expression level of your protein under different growing conditions and compare them to other proteins which you can select.

Figure 11. Transcription level of Spo0A(red), AbrB(dark blue) and Spo0F(light blue). Retrieved from: Subtiwiki (Zhu B & Stülke, 2018)

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BsubCyc

BsubCyc is part of the BioCyc database, here you will find features about the sequences, genes and proteins of Bacillus subtilis, it includes a genome browser, a regulatory overview of Bacillus subtilis general regulation, as well as individual and full metabolic maps.

Figure 12. Bacillus subtilis subtilis 168 metabolic overview. Taken from BsubCyc (Caspi et al. 2014)

Bacillus Genetic Stock Center

The Bacillus Genetic Stock Center (BGSC) is a genetic stock where you can find and order B. subtilis strains with specific phenotypes or modifications, as well as cloning vectors and bacteriophage for the genus Bacillus. According to their page, they have 1291 mutants derived from B. subtilis 168, 55 other strains derived from non-168 backgrounds, 54 Bacillus subtilis lysogens and 42 lytic phages of B. cereus, B. subtilis, and B. thuringiensis. This is the catalog of strains of B. subtilis: Bacillus Genetic Stock Center Catalog of Strains, Seventh Edition, Volume 1: Bacillus subtilis 168 . You can order directly from their page. We highly recommend you

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http://www.bgsc.org/_catalogs/Catpart1.pdf

http://www.bgsc.org/_catalogs/Catpart1.pdf

to search if the phenotype you need is found here since it will save you a considerable amount of work and the price is very accessible.

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Previous iGEM Bacillus subtilis Projects There is a list of iGEM projects developed with Bacillus subtilis on the parts registry ( http://parts.igem.org/Bacillus_subtilis#Ribosome_binding_sites ). However, this list only covers the competence until the year 2015. We believe that knowing what other teams have made in previous years is very important to come up with new and better ideas. Because of this we decided to include a list of iGEM projects related to B. subtilis from 2016 to 2019 to complement the information on the parts registry.

2016

Team Project title Description Link

UBonn Enzymatic Whitewashing - the

ecological approach to paper

recycling

The team developed a high throughput system that not only allows to quantify deinking efficiency but also to cheaply mass-produce enzymes using a Bacillus subtilis secretion system.

http://2016.igem.org/Team:UBonn_HBRS

Freiburg

Nanocillus-'cause spore is more!

After administration, conventional drugs are distributed throughout the whole body thus affecting both, diseased and healthy cells. By engineering the spores of probiotic Bacillus subtilis, a member of the human microbiome, the team established a low-cost carrier for well-tolerated treatment

http://2016.igem.org/Team:Freiburg

SVCE

LACTOSHIELD

The team developed a novel system that produces cationic antimicrobial peptides (cAMPs) to prolong the shelf life of milk by preventing bacterial contamination.

http://2016.igem.org/Team:SVCE_CHENNAI

Toulouse Paleotilis, a shield for the Lascaux

cave

The project consists of an engineered Bacillus subtilis strain which grows on bacterial organisms present in the cave. It releases antifungals when in close vicinity of fungi

http://2016.igem.org/Team:Toulouse_France

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http://parts.igem.org/Bacillus_subtilis#Ribosome_binding_sites













Groningen

CryptoGE®M: Encode it, Keep it

The world's silicon supply won't be able to cover the demand for flash data storage by 2040. The team’s goal was to safely send a key and an encrypted message in two separate spore systems of Bacillus subtilis.

http://2016.igem.org/Team:Groningen

UC Davis

CYANtific

In this project, the team demonstrates that the GAF domain of cyanobacteriochrome (CBCR) proteins are a viable natural alternative to artificial food dyes.

http://2016.igem.org/Team:UC_Davis

UofC Calgary The subtilis defence

The project is based on the administration of the naturally occurring peptide Bowman-Birk Protease Inhibitor (BBI), which has been shown to confer protection against DNA damage following radiation exposure. To express mBBI the team chose Bacillus subtilis.

http://2016.igem.org/Team:UofC_Calgary

2017


RDFZ-China Mobile Surfactant Factory Combating

Oil Spill With Engineered Bacillus

subtilis

The project engineered Bacillus subtilis that function as surfactin producing units to remediate contaminated soils. Biosurfactants and the introduction of Bacillus subtilis should have fewer impacts on soil microbiome and should be more effective than relying on bioremediation alone.

http://2017.igem.org/Team:RDFZ-China

Stanford-Brown

Mars: Getting there and staying there

The risk of mid-mission equipment failure, power shortages, or supply depletion incentivizes precautionary measures, but the financial strain of sending unnecessary mass into space limits this practice. Prioritizing repair over replacement, they were

http://2017.igem.org/Team:Stanford-Brown

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developing self-healing materials embedded with Bacillus subtilis. For longer-lasting energy, they were designing a 'biobactery' using linearly oriented E. coli to generate power. For renewable materials, they were engineering bacteria to synthesize and degrade rubber.

Sydney Australia

Designing Insulin that is Single-Chain

and Open-source (DISCO)

The project involved using synthetic biology to develop an insulin manufacturing system that is cost-efficient and simple, using the bacterial species Escherichia coli and Bacillus subtilis.

http://2017.igem.org/Team:Sydney_Australia

SZU-China CON-cure-CRETE

They designed a self-healing system for concrete. They used gerA as a biosensor for when liquid L-alanine present. They placed the spores of the Basilus subtilis into microcapsules along with nutrients and L-alanine powder. When the concrete cracks the tension will break the microcapsule and the water will infiltrate. The team designed a self-healing system for concrete. When there is a microcrack our system can be switched on and concrete can start to heal themselves.

http://2017.igem.org/Team:SZU-China

TMMU-China

Development of Quorum Sensing

Tool Kit for Gram-positive

Bacteria

The team wanted to develop a QS tool kit for Gram-positive bacteria. The tool kit is based on the Agr system from S.aureus, the PlcR-PapR system from Bacillus cereus, and the AimR-AimP system from the Bacillus subtilis bacteriophage Phi3T. The plan was to test the utility of this tool kit in Bacillus subtilis and Lactococcus lactis.

http://2017.igem.org/Team:TMMU-China

TU Dresden EncaBcillus It's a trap!

The project wants to introduce Peptidosomes as a new fundamental approach for generating and applying encapsulated bacteria. Using the powerful genetics of Bacillus subtilis

http://2017.igem.org/Team:TU_Dresden

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and its secretory capabilities we demonstrate communication and cooperation between separately encapsulated bacterial populations as well as the environment.

UIOWA

Development of a 3-Hydroxypropioni

c Acid Biosensor

The research project utilizes the 3-HP responsive genes found in P. putida and P. denitrificans as biological reporters which express luciferase in the presence of 3-HP. Then the system will be adapted to Bacillus subtilis.

http://2017.igem.org/Team:UIOWA

WPI Worcester

Go(a)t Lead? Bacterial Detection and Bioremediation

of Lead Contamination in

Drinking Water

Our project aims to improve lead testing and treatment by developing a lead biosensor and colorimetric lead assay, as well as a lead-binding probiotic.

http://2017.igem.org/Team:WPI_Worcester

2018


Goettingen

Glyphosate on my plate?! Detection

and inactivation of Glyphosate using

the soil bacterium Bacillus subtilis

The team’s aim is to engineer the Gram-positive model bacterium Bacillus subtilis for the detection and degradation of glyphosate.

http://2018.igem.org/Team:Goettingen

ICT-Mumbai SmartSoil: Rooting for Sustainable

Agriculture

The team studied changes in gene expression in the common soil bacterium, Bacillus subtilis, in response to root exudates of rice, wheat, tomato and soybean plants. The project is constructing a genetic amplifier using an exudate-inducible promoter to produce

http://2018.igem.org/Team:ICT-Mumbai

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phosphatase, which will help solubilize organic phosphate present in the soil.

ITESLA-Soundbio

Factor C The Difference: A

Synthetic Biology Alternative to the

LAL Endotoxin Detection Assay

The team sought to synthesize a codon-optimized sequence of Factor C and integrate it into Bacillus subtilis using a pAX01 backbone with a xylose inducible promoter. In the future, they hope to design a detection mechanism to signal for the cleavage of Factor C and the presence of endotoxin.

http://2018.igem.org/Team:iTesla-SoundBio

OLS Canmore Canada

The PET Peeve Project:

Bio-tagging PET Plastic for Efficient

Sorting and Recycling

The project uses synthetic biology to create a novel fusion protein that can specifically bio-tag PET plastic, so it can be sorted and recycled correctly. The project involves two proteins, PET hydrolase and a hydrophobin called BsIA, that are produced via a bacterial chassis called Bacillus subtilis.

http://2018.igem.org/Team:OLS_Canmore_Canada

SSTI-SZGD

Hyaluronic acid micro factory: a

bacterium produces low

molecular weight hyaluronic acid

The team constructed a recombinant strain Bacillus subtilis 168E which could directly produce different molecular weight HA products by regulating the activities of LHAase. They transferred the LHAase gene into Bacillus subtilis 168 which is from leech resources coding hyaluronidase. Therefore the HA could be enzymatically hydrolyzed to a different molecular weight.

http://2018.igem.org/Team:SSTi-SZGD

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UIOWA Investigating biosensors for the

industrial production of

3-hydroxypropionic acid

The research team transformed a promoter-regulator system that recognizes 3HP into Bacillus subtilis.


2019


BrownStanfordPrinctn

Towards an Astropharmacy

The team designed genetic templates to produce insulin, teriparatide, and hG-CSF using cellular systems to harnesses the speed of VmaxTM, the long-term viability of Bacillus subtilis, and production capability of E.coli, and commercial and lab-developed cell-free systems for their adaptability.

https://2019.igem.org/Team:BrownStanfordPrinctn

Duesseldorf

SynMilk- an eco-friendly

synthetic cow’s milk to save the

environment

The project consists in the production of the natural components of cow`s milk using methods from synthetic biology to modify microorganisms. The team modified Bacillus subtilis, Pichia pastoris, and the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 to produce the milk proteins heterologously.

https://2019.igem.org/Team:Duesseldorf

HZNFHS Hangzhou

Biological dinitrogen fixation

Nif-specific transcriptional

activator NifA gene modulates pH and

The team cloned the NifA gene from Sinorhizobium fredii, constructed the over-expression vector of pHT43 and transformed into Bacillus subtilis. The NifA over-expressed Bacillus subtilis modulated the soil pH from 4.0 to over 7.

https://2019.igem.org/Team:HZNFHS_Hangzhou

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bacteria around tea plants

Jiangnan-China

SUPERB The team used surfactin. In order to produce surfactin industrially, we modified Bacillus subtilis 168 by knocking out competition pathways, replacing promoters, and enhancing resistance efflux genes.

https://2019.igem.org/Team:Jiangnan-China

Orleans

The Metal`OSE Project (Optimized

Sludge Engineering)

The project aims to create a bacterium able to specifically remove heavy metals from sewage sludges and produce ethanol from the cellulose is contains.

https://2019.igem.org/Team:Orleans

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