iGEM 2023 — Team cambridge

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Notebook
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Lab Notebook

**Week 1 :** 27th Jul to 2nd Aug

#### Wet Lab :

1. Preparation of media and plates
2. Started cultures of *B. subtilis*

#### Human Practices :

1. Collected strains from Dr Mokherjee & learned protocols for culturing and transforming *B. subtilis*

**Week 2 :** 3rd Aug to 9th Aug

#### Wet Lab :

1. Optimising biomass staining assay
2. Preparing competent *B. subtilis* stocks

**Week 3 :** 10th Aug to 16th Aug

#### Wet Lab :

1. Optimising biomass staining assay
2. PCR for 5’ and 3’ HRs of ΔspoIIE construct
3. PCR to add overhangs to pMiniMAD (getting incorrect size band, unsure why)
4. Restriction digest to troubleshoot pMiniMAD issue
5. Preparation of electrocompetent *E. coli*

#### Dry Lab :

1. Introduction to HDock : Gathered subunits from AlphaFold databank for K-12 *E. coli* RNA polymerase (RNAP) - Played around with using the online software

**Week 4 :** 17th Aug to 23rd Aug

#### Wet lab :

- Optimising biomass staining assay
- Ordered new primers for cloning pMiniMAD
- Biosensor - shifting away from adrenaline, now focusing on expression of fusion protein

**Week 5 :** 24th Aug to 30th Aug

#### Wet Lab :

- Final optimisations for biomass staining assay
- Obtained correct size band for pMiniMAD with BbsI overhangs for digest & ligation

**Week 6 :** 31st Aug to 6th Sep

#### Wet Lab :

- Couldn’t get digest & ligation for ΔspoIIE construct to work, ordered new primers for Golden Gate
- Golden Gate with the new primers worked first time, confirmed we have the correct plasmid in our transformed *E. coli* by colony PCR
- Transformed miniprepped plasmid into *B. subtilis*, re-inoculating subcultures multiple times to remove resistance gene

**Week 7 :** 7th Sep to 13th Sep

#### Wet Lab :

- Cloning components for the engineered constructs from the genome of *B. subtilis*
- ΔspoIIE *B. subtilis* strain is ready, including resistance gene removed
- Doing biofilm formation assays and sporulation assays with the ΔspoIIE strain
- Designed biocontainment system & ordered primers/DNA synthesis
- *E. coli* biosensor transformant is ready, checking fluorescence in plate reader and with fluorescence microscopy

**Week 8 :** 14th Sep to 20th Sep

#### Wet Lab :

- Cloning components for the engineered constructs from the genome of *B. subtilis*
- Long-term biomass assays with ΔspoIIE
- Spore CFU count assay – no colonies grew even at only 10-3 dilution, likely due to issues with the FeSO4 in the lab being oxidised; ordered new FeSO4
- Oligo annealing for RBS and terminator sequences
- Did Golden Gate with synthesised biosensor construct, which failed; the oligo annealed terminator is the most likely issue; re-did oligo annealing with thermal cycler (instead of heat block)
- Golden Gate assembly with oligo annealed RBS and terminator failed; ordered new primers
- Did genomic extraction from *B. subtilis* to clone some large fragments that we’re struggling to clone from colony PCR

**Week 9 :** 21st Sep to 27th Sep

#### Wet Lab :

- Cloning components for the engineered constructs from the genome of *B. subtilis*
- Spore CFU count assay with new FeSO4 showed ΔspoIIE was effective
- Golden Gate assembly with oligo annealed RBS and terminator failed; diagnostic PCR and restriction digest suggests the oligo annealed parts are causing the issue (unsure why); ordered new primers to clone the RBS and terminator from the genome/distribution kit
- Testing/optimising the pathogenic biofilm incidence assay with wild type *B. subtilis* 3610

**Week 10 :** 28th Sep to 4th Oct

#### Wet Lab :

- Cloned RBS and terminator sequence from the genome/distribution kit
- Golden gate with these RBS and terminator parts finally worked; assembled and transformed the constructs into *E. coli*, then into *B. subtilis*
- Took confocal microscopy images of wild type biofilm; ordered microscopy dishes to look at the biofilms of the engineered strains & invasion of mVenus-expressing *E. coli*
- Biofilm formation assays on stainless steel and PVC

**Week 11 :** 5th Oct to 11th Oct

#### Wet Lab :

- Assembled and transformed the final few constructs
- Biofilm biomass assays of engineered constructs
- Pathogenic biofilm incidence assays with engineered constructs
- Inducible biofilm formation assays with mstX construct
- Did confocal microscopy with the engineered strains – unsuccessful, possibly due to user error or issues with the microscopy dishes

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Engineering
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* [Introduction](#1)
* [Sporulation K/O](#2)
* [Formation Rate](#3)
* [Biomass Increase](#4)
* [Seaweed Utilisation](#5)
* [Pathogen Biosensor](#6)
* [Biocontainment](#7)

## Introduction

We decided to utilise *B. subtilis* as the chassis for our protective biofilm, due to the ability of *B. subtilis* to naturally form protective biofilms on plant roots, in addition to it being classified as a generally recognized as safe organism with no known pathogenic strains. Whilst there is some literature suggesting that wild type *B. subtilis* biofilms can be effective at reducing pathogen incidence and mitigating microbially induced corrosion, a field study testing Bacillus and Pediococcus protective biofilms in broiler chicken farms showed that they seem to lack long-term efficacy, possibly due to the identified limited surface coverage (Guéneau *et al.*, 2022). Additionally, *B. subtilis* biofilms normally disperse and form spores during prolonged nutrient stress. Therefore, we aimed to engineer a *B. subtilis* strain that can form more effective protective biofilms, by designing and integrating constructs into the genome to achieve the following:

1. Non-Sporulating Biofilm
2. Increased Biofilm Formation Rate
3. Increased Biomass of Mature Biofilm
4. Inducible Biofilm Formation and Orthogonal Nutrient Utilisation
5. Incorporated Pathogen Biosensor
6. Incorporated Biocontainment Mechanisms

## 1. Non-sporulating Biofilm

**Reason:**
 From our meeting with Dr Diana Fusco, we learned that a major limitation of *B. subtilis* as a protective biofilm is that *B. subtilis* biofilms disperse and sporulate under prolonged nutrient stress; hence, wild type *B. subtilis* may not provide effective long-term surface coverage.

**Research:**
As sporulation and biofilm formation share significant upstream regulatory pathways, it was important that we targeted a knock-out candidate sufficiently downstream in the sporulation pathway to not affect biofilm formation; however, targeting a component too far downstream may result in cells that are committed to the spore state, but are unable to proceed and hence simply die. From Gray (2016), we identified *spoIIE* – a phosphatase required for correct asymmetric division during sporulation – as a promising knockout candidate to meet these criteria.

Design
To knock out *spoIIE*, we utilised homologous recombination in *B. subtilis* by designing a construct containing ~1,000 bp homology regions flanking either side of the *spoIIE* gene, to be integrated into the genome of *B. subtilis*.

![Sporulation K/O](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/sporulation-ko.png)

Build
We assembled both spoIIE homology regions into the pMiniMAD backbone, utilising Golden Gate assembly. The construct was then transformed into *E. coli* and miniprepped before transformation into *B. subtilis* by homologous recombination.

Test
 The ΔspoIIE strain showed almost complete loss of spore formation, exactly as hoped. ΔspoIIE also formed a mature biofilm significantly faster than wild type *B. subtilis* and appeared to have slightly greater total biofilm biomass overall; hence, there was no notable negative effect on biofilm formation, and instead a significant benefit to biofilm formation was identified.

Learn
From our meeting with Dr Briandet, he informed us that commercial *B. subtilis* probiotics are commonly distributed as spores, due to their increased stability; hence, whilst knocking out sporulation may increase long-term stability and increase biofilm formation, this may limit the commercial viability of B.Max.

**Improve:**
Based on Dr Briandet’s advice, we designed a construct for genome integration of *spoIIE* under an inducible promoter; in theory, this could be integrated into a ΔspoIIE strain to allow for inducible sporulation in the presence of the inducer during culturing to allow for spore harvesting for commercial distribution, but during application, in the absence of the inducer, the biofilm would be non-sporulating.

![Inducible Sporulation Construct](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/inducible-sporulation.png)

Additionally, as knocking out *spoIIE* from the sporulation genetic pathway resulted in significantly increased biofilm formation rate, we designed constructs to modify genes within the biofilm genetic pathway to hopefully achieve a similar outcome.

## 2. Increased Biofilm Formation Rate

**Reason:**
 Based on field studies of protective *Bacillus* and *Pediococcus* biofilms in broiler chicken farms, these protective biofilms seemed to lack long-term efficacy, possibly due to the identified limited surface coverage (Guéneau *et al.* 2022). Hence, increased biofilm formation rate may help increase surface coverage, providing a more effective protective biofilm than non-engineered strains. Additionally, increased biofilm formation rate should allow for the protective biofilm to form quicker, providing less time for pathogens or corrosive microbes to take up the empty space following cleaning.

**Research:**
We created a gene regulatory model for biofilm formation in *B. subtilis*, based on the data in published literature. From this model, we identified that increasing the concentration of sinI, abbA, or remA may result in increased rate of biofilm formation.

Design
We designed constructs for expression of the regulatory genes *sinI*, *abbA*, and *remA* under the control of a xylose inducible promoter (BBa\_K733002), with each construct construct flanked by homology regions for subsequent integration into the genome of *B. subtilis*.The inducible promoter should allow for testing the effect of different levels of gene expression by varying the inducer concentration.

![Sporulation K/O](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/8-component-part.png)

Build
Each construct was assembled in an 8-part Golden Gate assembly. Correct size bands were identified for each construct by colony PCR following transformation into *E. coli*, however only *sinI* was successfully subsequently integrated into the genome of *B. subtilis* 3610 in the time we had available.

Test
 Biofilm biomass safranin staining assays confirmed that increased production of SinI results in significantly increased rate of biofilm formation. There was no significant difference between the induced and uninduced condition (with and without 0.5% xylose), suggesting that the BBa\_K733002 xylose inducible promoter is significantly leaky.

Learn
 Due to difficult troubleshooting an 8-part Golden Gate assembly, in the future we would design this construct with fewer parts. With more time, we would also like to test the effect of increased expression of *abbA* and *remA*.

## 3. Increased Biomass of Mature Biofilm

**Research:**
From Bassler *et al.*(2015) and McKenzie *et al.* (2022), we identified that the density and thickness of the biofilm extracellular matrix appears to play a role in preventing pathogen invasion. Hence, increasing total biomass (and increasing density and thickness) may result in B.Max having increased resistance to invasion.

From our gene regulatory network model of biofilm formation, it seemed that increasing expression of the signalling genes may also result in increased total expression of the biofilm associated genes, hence resulting in increased total biomass. Additionally, we considered increasing the expression of the major polysaccharide and protein components of the biofilm matrix, the eps and tapA-sipW-tasA operons, in addition to ypqP gene from *B. subtilis* NDmed (involved in strong biofilm formation, disrupted in common lab strains of *B. subtilis*, such as 3610, likely involved in synthesis of extracellular polysaccharides) (Sanchez-Vizuete *et al.* 2015).

Design
 Designed constructs consist of eps operon, tapA-sipW-tasA operon, and ypqP gene under the control of a xylose inducible promoter (BBa\_K733002), with the whole construct flanked by homology regions for subsequent integration into the genome of *B. subtilis*. The inducible promoter should allow for testing the effect of different levels of gene expression by varying the inducer concentration.

Build
Each construct was assembled in an 8-part Golden Gate assembly. Correct size bands were identified for the tapA-sipW-tasA operon and ypqP gene by colony PCR following transformation into *E. coli*, however only the tapA-sipW-tasA operon was successfully subsequently integrated into the genome of *B. subtilis* 3610 in the time we had available.

Test
Biofilm biomass safranin staining assays confirmed that increased expression of the tapA-sipW-tasA operon results in significantly increased biofilm formation, in addition to increased expression of the regulatory gene *sinI* and the downstream gene *mstX* (discussed in the following section).

![Biofilm Biomass Assay](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/biofilm-biomass.png)

Learn
 Due to difficulty troubleshooting an 8-part Golden Gate assembly, in the future we would design this construct with fewer parts. With more time, we would also like to test the effect of increased expression of the eps operon and ypqP.

## 4. Inducible Biofilm Formation and Orthogonal Nutrient Utilisation

**Reason:**
As we want to increase total biomass of the protective biofilm, we considered that nutrient usage/availability may be a limiting factor in biofilm formation. Published literature suggested that *B. subtilis* biofilms primarily form under low nutrient conditions and hence biofilm regulatory pathways may not be sufficiently active under high nutrient conditions. Inducible biofilm formation may allow for biofilm formation under higher nutrient conditions, and hence possibly increased total biofilm biomass.

Additionally, given that surface coverage appears to be a major limitation of protective *B. subtilis* biofilms, it is possible that inducing biofilm formation prior to application may result in significantly increased adhesion, due to production of the sticky extracellular matrix components, resulting in improved surface coverage.

**Research:**
Choe *et al.* (2013) demonstrated that overexpression of the *mstX* gene resulted in activation of KinC (which normally can act as a sensor for low nutrient conditions), resulting in the induction of the biofilm formation pathways.

Design
The designed construct consists of the mstX gene under the control of a xylose inducible promoter (BBa\_K733002), with the whole construct flanked by homology regions for subsequent integration into the genome of *B. subtilis*.

Build
 The mstX construct was assembled in an 8-part Golden Gate assembly. Correct size bands were identified by colony PCR following transformation into *E. coli*, and correct size bands were identified by colony PCR in *B. subtilis*, hence integration into the *B. subtilis* genome was successful.

Test
Biofilm biomass safranin staining assays identified that increased expression of mstX appears to result in significantly increased rate of biofilm formation.
 Inducible expression of *mstX* also resulted in significantly increased biofilm formation under high nutrient conditions (left = biofilm formation of mstX overexpressing *B. subtilis* 3610 (in pink) and wild type *B. subtilis* 3610 on standard MSgg media, right = biofilm formation of *mstX* overexpressing *B. subtilis* 3610 (in pink) and wild type *B. subtilis* 3610 on 4x nutrients containing MSgg media)

![Biofilm Biomass Assay](https://static.igem.wiki/teams/4769/wiki/general-images/image1.png)

Learn
 Whilst overexpression of mstX seems to increase rate of biofilm formation, we considered that even if nutrient utilisation was normally limiting, providing external nutrients to an environment such as farm surfaces would also provide these nutrients to pathogens or corrosive microbes. Hence, ideally we would be able to apply an external nutrient source that our protective biofilm could digest/utilise, but pathogens and corrosive microbes could not.

**Improve:**
Shepherd *et al.* (2018) identified a ~40-60 kb region within the genome of Bacteroides plebeius/Phocaeicola plebeius that provides the ability to digest the polysachharide, porphyran, found in seaweed. We designed constructs to integrate the minimum required ~40 kb of this construct into the genome of *B. subtilis* by homologous recombination of 6 ~7 kb fragments, utilising 2 different resistance genes at the 3’ end that would be deleted from the genome by homologous recombination of the subsequent fragment, due to the 5’ homology region being homologous to the region upstream of this resistance gene.

## 5. Pathogen biosensor

**Reason:**
 Although B.Max is designed to minimise pathogen invasion, it inevitably would not be completely effective. An incorporated pathogen biosensor for major foodborne pathogens may allow for targeted cleaning/use of biocides if necessary.

**Research:**
 When scavenging across the literature on the molecular signals that enteric pathogens produce, we came across autoinducer-3 (AI-3). AI-3 is used by enteric pathogens to sense and signal that they are in the intestinal environment, and is sensed by the two-component system (TCS) QseBC. Hence we chose this system as the base of the biosensor due to AI-3’s heavy involvement in the virulence of enteric pathogens. In the TCS, upon binding of AI-3 by QseC, QseC phosphorylates QseB. QseB-P will then bind onto a promoter and activate the transcription of the genes downstream, for example a GFP so that the system acts as a biosensor for AI-3.

Design
 Our final construct for the AI-3 biosensor was designed as below (BBa\_4769605):

![eng1](https://static.igem.wiki/teams/4769/wiki/engineering/eng1.png)
The QseB and QseC genes are controlled under pVeg, a constitutive promoter, so that *B. subtilis* would be able to receive AI-3. A superfolder GFP gene is downstream of the QseB-activated promoter. By not placing a terminator after the sfGFP, QseBC would also be expressed after AI-3 reception, hence increasing *B. subtilis*’ sensitivity to AI-3 and increase the fluorescence produced. The gene is inserted into *B. subtilis* using pMiniMAD with the homology regions of AmyE.

We have decided to test our idea on *E. coli* DH5α as a proof of concept. To do so, we have built a construct consisting of the QseB-activated promoter (BBa\_K4769600) which we extracted from the *E. coli* genome, a T7 g10 RBS (BBa\_K4769607), a superfolder GFP which we amplified from pJUMP28-1A(sfGFP) (BBa\_J428353), and a conventional terminator (BBa\_J428091). We have selected AE\_lacZ pDEST (BBa\_J435320), a high copy number expression plasmid, as the backbone. This construct assumes QseB and QseC are expressed from the *E. coli* genome.

Build
We have assembled our constructs using Golden Gate Assembly and transformed them into *E. coli* DH5α and *B. subtilis* respectively.

Test
In the DH5α strain containing the proof of concept plasmid, the transformed bacteria doesn’t fluoresce, in contrast to what is expected as QseBC/AI-3 is a quorum sensing system and the AI-3 concentration should increase as cell density increases.

Learn
We went back to literature and found evidence showing that AI-3 expression is linked to AI-2 quorum sensing, and AI-2 is not produced in the DH5α strain, which may contribute to the fact that our transformant doesn’t fluoresce. A method to overcome this is to directly add AI-3, or its analog adrenaline, to the media and measure the fluorescence. However, adrenaline cannot be obtained in our lab due to the risk involved, hence we decided to switch our proof of concept to proving that QseC can be properly incorporated into the plasma membrane of *B. subtilis*, as QseC is only present in gram-negative bacteria and is usually inserted into the inner membrane.

Moreover, we identified another potential reason for the construct to fail, where upon inspecting the genome of DH5α, an araC family transcriptional repressor is present in front of the QseBC genes. As our construct assumes the genomic expression of the two proteins, the repressor could be problematic.

We also ideated a protocol which doesn’t require adrenaline as a replacement for AI-3. To do so, we would culture an AI-3 producing bacteria, then centrifuge it to obtain the AI-3 containing supernatant, which can be added to our transformant that is cleaned with 10% glycerol.

**Improve:**
We have hence designed a fusion protein between QseC and sfGFP (QseC-sfGFP), using the linker sequence GSAGSAAGSGEF (Waldo *et al.*, 1999) to connect both proteins. An Alphafold prediction of the structure shows a stable sfGFP can be formed.

![eng1](https://static.igem.wiki/teams/4769/wiki/engineering/eng2.png)

To overcome the araC family repressor problem, we have also designed a construct for *E. coli* that includes the QseBC coding genes. The construct is assembled in a similar fashion as to the *B. subtilis* biosensor, but with Pveg replaced by J23100. (BBa\_J23100), a standard constitutive promoter, and the *B. subtilis* RBS replaced with T7 g10-L RBS for *E. coli*.

Test
Unfortunately we didn’t have the time to transform the QseC-sfGFP construct into *B. subtilis*. However, if given the time, a successful localisation would have fluorescence around the periphery of the cell.

## 6. Biocontainment

**Reason:**
 Whilst *B. subtilis* is generally recognised as safe and is already used as a probiotic for humans, plants, and animals, it is important that our engineered strain does not end up where it was not designed to be and possibly cause unintended consequences. Additionally, although our engineered strain is designed to minimise the probability of invasion, over time there is the possibility of these beneficial biofilms being invaded by pathogenic or corrosive microorganisms, which may then be difficult to remove during cleaning. Hence, we recognised the need for biocontainment to prevent unintended spread of B.Max, in addition to the need for inducible biofilm disruption (self-destruction) to allow for effective cleaning of any invading microbes.

Design - Preventing Unintended Spread

To prevent the unintended spread of B.Max, we considered 3 of the major biocontainment mechanisms – a toxin-based kill switch that results in toxin expression and cell death under certain condtions, engineered auxotrophy that only allows cells to survive in the presence of supplemented nutrients, or conditional expression of essential genes under certain conditions. As B.Max may be broadly applied over large surfaces with varying nutrient compositions, engineered auxotrophy did not appear to be reasonable in this case. Additionally, when considering the long-term evolutionary stability of the biocontainment mechanism (i.e. how easy it is to break it), toxin-based kill switches appear significantly less evolutionarily stable (i.e. easier to break) than conditional essential genes. Hence, we decided to utilise conditional essential genes for this biocontainment mechanism.

As B.Max is intended to survive only in the biofilm-associated state, we decided to design a biocontainment mechanism that allows biofilm-associated cells to survive, but results in the death of planktonic cells. To do so, we considered utilising the biofilm master regulator in *B. subtilis*, SinR – SinR binds to an operator region upstream of the promoter of genes involved in biofilm formation, repressing expression of these genes during the planktonic state. During the planktonic-to-biofilm transition, SinR is sequestered by SinI, preventing binding of SinR to the operator regions and allowing for expression of biofilm-associated genes. Hence, adding a SinR-binding operator region upstream of the promoter of an essential gene may allow for the desired biocontainment mechanism of survival in biofilm-assocaited state and death in planktonic state.
![](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/biocontainment1.png)

However, a single SinR-binding operator region upstream of the promoter of a single essential gene also appears evolutionarily unstable (i.e. it could be easily broken by disruption of the SinR-binding operator region). To increase the evolutionary stability of this biocontainment mechanism, we considered adding multiple binding sites for SinR, and binding sites for other transcription factors involved in biofilm formation, upstream of the promoters of multiple essential genes. From this, the probability of all binding sites being disrupted for all of the conditional essential genes would appear to be very low.

As *B. subtilis* would be planktonic during culturing and application, in this biocontainment system the cells would die prior to being able to form a biofilm. Hence, we considered inserting an additional copy of the essential gene at a different site in the genome, expressed under an inducible promoter. This would effectively form an AND gate for survival, where cells have to either be in the presence of an inducer (which could be provided during culturing) or in the biofilm-associated state.

To identify the optimal essential genes to conditionally express, we analysed the BsubCyc database for every essential gene in *B. subtilis* to identify those which have annotated promoter regions supported by experimental evidence and contain only a single gene under the control of that promoter (to avoid unintentionally making other genes conditional). From this, we identified gyrA, gltX, and rpsD as promising candidates.

For designing the operator region, we decided to utilise the naturally-occuring operator regions upstream of the eps and tapA-sipW-tasA operons (encoding the major polysaccharide and protein components of the biofilm matrix, respectively). These operator regions are well-documented based on experimental evidence, hence it was possible to design constructs to insert these operator regions upstream of the promoter of the essential genes, with the same distance between the operator elements and transcription start site that occurs in the eps and tapA-sipW-tasA operons.
![](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/biocontainment2.png)

(Images from BsubCyc)

Build
 Each of the three essential genes were assembled as an 8-part assembly by Golden Gate assembly, under the control of a xylose inducible promoter (BBa\_K733002), between homology regions for subsequent integration into the genome of *B. subtilis*. However, due to the xylose inducible promoter being significantly leaky, it was not possible to achieve no expression of the essential genes and hence there was no point proceeding with introducing the operator regions upstream of essential genes.

Design - Inducible Biofilm Disruption
To allow for effective removal of B.Max and any invading microbes during cleaning, we considered the optimal mechanisms for inducible biofilm disruption. Whilst there is data supporting biofilm disruption by application of the DNase, DNase I, and the protease, proteinase K, these are eukaryotic proteins which are unlikely to function optimally in *B. subtilis*. Instead, we considered other proteins, such as the bacterial DNase, NucB, the bacterial proteases, NprB and Bpr, and the bacterial racemase, YlmE. Expression of some combination of these proteins following application of an inducer may result in significant amounts of biofilm disruption to allow for effective cleaning.

Build
The biofilm-degrading enzymes were each assembled as an 8-part assembly by Golden Gate assembly, under the control of a xylose inducible promoter (BBa\_K733002), between homology regions for subsequent integration into the genome of *B. subtilis*. However, we were unfortunately not able to correctly assemble the constructs in the time we had available.

![](https://static.igem.wiki/teams/4769/wiki/general-images/engineering/engineering/biocontainment3.png)

Learn
 Due to difficulty troubleshooting an 8-part Golden Gate assembly, in the future we would design this construct with fewer parts. With more time, we would also like to test the effect of inducible expression of the biofilm-degrading enzymes on degradation of mature biofilms.

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{% block design %}
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Results
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![Title Banner](https://static.igem.wiki/teams/4769/wiki/bannerimages/banner-summer-afternoon.png)
![Title Banner](https://static.igem.wiki/teams/4769/wiki/bannerimages/banner-results2.png)

* [Sporulation Knockout](#1)
* [Biofilm Engineering](#2)
* [Invasion Resistance](#3)
* [Surface Growth](#4)
* [Confocal Growth](#5)
* [Biosensor](#6)

## Sporulation Knockout

One of the main aims of our project was to knock out sporulation in *B. subtilis* to provide a longer lasting biofilm. We aimed to achieve this by knocking out the spoIIE gene in *B. subtilis*, which encodes a phosphatase that plays a major role in spore formation.

To test if the ΔspoIIE strain successfully knocked out sporulation, we performed a spore count assay, where cells are cultured in starvation media to induce sporulation, followed by heating some of the samples at 70 °C to kill any cells that are not spores. The number of spores and the number of vegetative cells can then be determined by serially diluting and plating on LB agar to count the colony forming units (CFUs).

The below figure shows the number of vegetative cells and spores for wild type *B. subtilis* 3610 and ΔspoIIE *B. subtilis* 3610 – these results show almost a total loss of spore formation in the ΔspoIIE strain: around 1 in 4 of the wild type culture are spores, whereas only 1 in 20,000 of ΔspoIIE culture are spores!

![](https://static.igem.wiki/teams/4769/wiki/general-images/spore-count2.png)

As the goal of knocking out sporulation was to increase the long-term stability of the biofilm, we also wanted to check how the biofilm biomass of ΔspoIIE compared to wild type *B. subtilis* over time. For this, we optimised a biofilm biomass assay by staining with safranin – as seen below, there does not appear to be significant amounts of biofilm dispersal/breakdown in wild type by 2 weeks (likely due to these assays being carried out in TSB, a nutrient rich media), so from this data we cannot conclude if ΔspoIIE is less affected by biofilm dispersal; however, ΔspoIIE appears to form a mature biofilm significantly faster than wild type *B. subtilis* and appears to have slightly greater total biofilm biomass overall, hence also achieving our aim of increased biofilm formation

![](https://static.igem.wiki/teams/4769/wiki/general-images/biomass2.png)

## Increased Biofilm Formation

To achieve increased biofilm formation, in addition to ΔspoIIE, we designed constructs for inducible expression of multiple regulatory components of the *B. subtilis* biofilm formation pathway, in addition to inducible expression of the major protein component of the biofilm matrix.

We tested the biofilm formation of these engineered strains in the presence of the inducer, xylose, using the same safranin stain biofilm biomass assay. These results showed that overexpression of the genes *sinI*, the *tasA*> operon and *mstX* all result in significantly increased biofilm formation rate, compared to wild type *B. subtilis*.

![](https://static.igem.wiki/teams/4769/wiki/general-images/24-hr-biomass2.png)

## Decreased Pathogenic Biofilm Formation

We then wanted to confirm that protective biofilms can indeed reduce the incidence of surface attached pathogenic or corrosive microbes. To do so, protective *B. subtilis* biofilms were first formed in wells of 96-well plates, followed by introduction of a model pathogen – a strong biofilm-forming strain of non pathogenic *E. coli* containing an ampicillin resistance plasmid – into wells with or without these protective biofilms; after 24 hours, the number of surface-attached cells in wells with or without the protective biofilms were determined by washing, scraping the surface of the wells, and CFU counting on ampicillin plates.

The below results show that the protective biofilms significantly reduced the incidence of pathogenic biofilms, by around 100,000 fold. Additionally, our data shows that B.Max appears to be even more effective at preventing pathogenic biofilms than wild type *B. subtilis*.

![](https://static.igem.wiki/teams/4769/wiki/general-images/cfu-count2.png)

## Biofilm Formation on Common Farm Surfaces

Whilst all of our previous experiments were performed in polystyrene plates, it is important that the protective biofilms can also form on surfaces commonly found in farms. Based on our meeting with Dr Briandet, we learned that common materials used in agriculture include stainless steel and PVC. Therefore, we 3D printed plastic wells which were adhered to the surfaces of stainless steel and PVC sheets; *B. subtilis* cultures were then introduced into the wells as in previous assays, and after 72 hours of incubation the the media was removed, the wells were washed, and biofilm formation was identified visually.

These assays show that *B. subtilis* can indeed form biofilms on both stainless steel and PVC (stainless steel on left, PVC on right). Hence, *B. subtilis* seems an appropriate choice for applications of protective biofilms in agriculture.

![](https://static.igem.wiki/teams/4769/wiki/general-images/surface-growth.png)

## Confocal Imaging of Biofilms

Additionally, during our meeting with Dr Briandet, he advised that confocal microscopy could be a valuable technique for visualising the ultrastructure of the biofilm. Whilst his lab and seemingly most papers in the literature utilise fluorescent stains such as Syto 9, these are fairly expensive and not available to many iGEM teams. Therefore, we tested confocal imaging utilising a strain of *B. subtilis* 3610 expressing a red fluorescent protein (mScarlet, kindly provided by Dr Diana Fusco). We found that this was effective for viewing mature biofilms grown in glass bottom microscopy dishes, as shown below by the 3D image of a mature *B. subtilis* biofilm captured by confocal microscopy.

![](https://static.igem.wiki/teams/4769/wiki/general-images/biofilm-viewer.gif)

Whilst we hoped to utilise confocal microscopy to investigate the biofilm ultrastructure of our engineered strains, we were unfortunately unable to do so in the time we had available.

## Biosensor

We successfully assembled and transformed an AI-3 sensing construct into *E. coli* DH5α, however since DH5α likely does not produce AI-3, the transformant did not produce any detectable fluorescence. We later changed our focus to identifying if QseC can be expressed and localised to the plasma membrane of *B. subtilis*. We created a QseC-sfGFP fusion protein and confirmed the size of the construct with gel electrophoresis, however we unfortunately did not have enough time to transform it into *B. subtilis* to visualise the localisation.

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