iGEM 2022 — Team lund

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{% block title %}Notebook{% endblock %}
{% block lead %}Follow our journey through the lab{% endblock %}
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Here, you can find the detailed structure of our every day lab life. We invite you to spend some time following our highs and lows through the experimental phase of our project.

| Week | Link |
| --- | --- |
| 23 | [Entry for week 23](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/23.pdf) |
| 24 | [Entry for week 24](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/24.pdf) |
| 25 | [Entry for week 25](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/25.pdf) |
| 26 | [Entry for week 26](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/26.pdf) |
| 27 | [Entry for week 27](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/27.pdf) |
| 28 | [Entry for week 28](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/28.pdf) |
| 29 | [Entry for week 29](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/29.pdf) |
| 30 | [Entry for week 30](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/30.pdf) |
| 31 | [Entry for week 31](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/31.pdf) |
| 32 | [Entry for week 32](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/32.pdf) |
| 33 | [Entry for week 33](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/33.pdf) |
| 34 | [Entry for week 34](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/34.pdf) |
| 35 | [Entry for week 35](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/35.pdf) |
| 36 | [Entry for week 36](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/36.pdf) |
| 37 | [Entry for week 37](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/37.pdf) |
| 38 | [Entry for week 38](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/38.pdf) |
| 39 | [Entry for week 39](https://static.igem.wiki/teams/4373/wiki/sponsors/notebook-entries/39.pdf) |

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{% block title %}Engineering Success{% endblock %}
{% block lead %}The three engineering cycles of Beecilli{% endblock %}
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![](https://static.igem.wiki/teams/4373/wiki/engineering/success.png)

# Cycle 1 - Cloning

## Design

---

With a traditional cloning approach based on restriction and ligation, the fragments were flanked with vector specific cloning sites. The choice of cloning sites was determined by their presence within the vectors. With multiple sites present inside the gene of interest (GOI), the corresponding nucleotides were altered. Each GOI was synthesised with one 5’-HindIII and two restriction sites on the 3'-end, SalI and NheI.

Two strategies for recombinant protein production were followed – chromosomal integration and plasmid expression. Each vector carries specific functional units. However, to reach high-copy plasmids and thus increase transformation efficiency later in *B. subtilis*, an extra cloning step in *E. coli* was introduced. In order to achieve replication in *E. coli*, an *ori* was integrated into these vector. For further positive selection, antibiotic resistance was considered. Here, pCW7 and pCW101 transformants were selected under chloramphenicol and ampicillin pressure.

## Build

---

Bringing our designed fragments to the next level and thus into the vector backbone, the synthesised inserts were digested with HindIII and NheI or SalI. These blunt ends were also introduced to both vectors creating two linear backbones utilised for consequent cloning into pCW101 or pCW7. T4 Ligase joined plasmids were further uptaken by competent *E. coli* cells producing high numbers of recombinant transformants.

## Test

---

Screening for successful clones was performed via Colony PCR. Here, newly designed primers bind within the vector backbone, thus outside of the insert amplifying the correctly integrated fragment. Fragments with the correct sizes were obtained for goxB-hysp and goxB-ylb inserts [(See results)](https://2022.igem.wiki/lund/results). Their plasmids were extracted and sequence analysed by an external provider.

## Learn

---

The traditional cloning method did allow us to successfully clone two gene fragments into pCW101 vectors. However, tenacious adaptation of protocols in terms of digestion conditions and extreme insert to vector ratios (up to 30:1) were needed to gain the desired product. PCR analysis of the ligation mixture gave us further insight, that the cloning problem lies within insufficient ligation and not transformation. Further we sequenced the digested fragments to enable verification of correctly synthesised DNA from IDT. Sequencing results confirmed the assumed homology. Nevertheless, it was not possible to obtain any other recombinants.

## Re-design

---

To reach higher cloning efficiency, an exonuclease based method, called Gibson Assembly©, was utilised. By creating homologous overhang regions at the desired insert via simple PCR, the following one-tube reaction allowed sufficient cloning. Through this method, transformants for pCW101-*goxB*-veg and pCW7-*goxB*-ylb were realised. However, only the former carried the correct sequence. Difficulties in amplifying the *goxA* or *pnGHIJKL* fragments did not allow for further alignment reaction. Redesign of overhang primers should resolve this obstacle. Nevertheless, with the intention of moving the project quickly towards to the next phase, after acquiring *goxB* constructs for all three promoters, the focus was shifted away from goxA and *pnGHIJKL* and no further cloning effort was attempted. The obtained recombinants however provide proof for a successful undergone engineering cycle.

# Cycle 2 - Protein Production

## Design

---

Design of a protein expression strategy mainly followed safety concerns. As our probiotic will be liberated into the environment, undesirable protein production could create glyphosate-resistance in non-target species. Therefore fragments carrying our gene of interest (GOI) under stationary phase promoter Pylb were designed. Furthermore, constant levels of glyphosate degrading proteins are beneficial as we cannot predict, when bees come into contact with the herbicide, thus the constitutive promoter Pveg was introduced upstream of the GOI. An IPTG-inducible promoter Physp was also analysed to provide general proof of degradation activity.

## Build

---

To avoid constant selective pressure in connection to plasmid stability, chromosomal gene expression was here the favoured strategy. However, homologous recombination requires a common set of integration sites present in target chromosomal DNA and plasmids. Here, pCW101 inhabits *amyE* integration sites generally used for chromosomal integration in *B. subtilis*. By transformation of the generated pCW101-recombinants into our expression host, *goxB* under different promoters was integrated into its DNA thus building the foundation for a stable protein producing system.

## Test

---

The correct integration was confirmed with a starch-based amylase test (See protocols). Following protein expression experiments were performed in minimal media supplemented with glucose and glyphosate. Cultivation at these conditions should provide insight if goxB will be produced during external stress triggered by glyphosate. Samples were analysed via SDS page [(See results)](https://2022.igem.wiki/lund/results).

## Learn

---

No proteins corresponding to goxB could be detected here. By change of cultivation conditions such as decreased incubation temperature an altered outcome was expected. Furthermore, cultivation in minimal media might not provide optimal conditions for sufficient growth. However, goxB was neither identified after cultivation at altered temperatures nor at growth on LB media. Sequence analysis of the integrated promoter was supposed to give further insight. However, due to difficulties with the extraction of sufficient amounts of chromosomal DNA, screening for potential mutations was not possible.

## Re-design

---

By change of detection method from SDS to more sensitive western blot, the presence of our target protein might be defined. Furthermore, with this method, we could analyse highly diluted supernatants for unlikely protein secretion. However, the availability of antibodies with high specificity towards goxB was limited and did not allow us further investigations. Nevertheless, high level protein expression might be generally suppressed. The *lac* operon is still present inside the vector when inserts are introduced. Potential *lac* repressor binding sites within the promoter regions of Pveg and Pylb might contribute to inhibited protein expression. By deleting the *lac* repressor, this eventual effect might be eliminated. However, one should screen for possible binding sites within the promoter region first. Claes von Wachenfeldt further pointed out that protein expression can alter between spore-forming and non-spore-forming *Bacillus* strains[1](#ref-1). Therefore characterising the level of protein expression in *Bacillus subtilis* 168, a well described spore-former, was considered. However, due to high risk of spore contamination, the laboratory safety officer did not allow us to work with this variant.

# Cycle 3 – Glyphosate detection

## Design

---

By measurement of glyphosate decline in the supernatant, the activity of the introduced glyphosate dehydrogenase should be determined. Described detection methods are based on spectrophotometric analysis. As glyphosate does not carry chromophoric groups making it visible under UV, one such molecule had to be introduced by derivatization. In the chosen method, we used FMOC-Cl which binds to glyphosate’s amino-group and thus enabling absorbance around 265 nm[2](#ref-2).

## Build

---

Glyphosate generally blocks the production of aromatic compounds. By expression of our heterologous protein, this effect should be strongly inhibited. Analysis of glyphosate degradation was therefore assessed in media without additional aromatic amino acids. The amount of supplemented glyphosate concentration was determined based on MIC assay results. Although all constructs, besides goxB-veg, had a MIC of 500 mg/L after 5 day incubation, we decided to perform further degradation experiments with lower amounts as time being the limiting factor. With all *Bacillus* variants growing after 24 hours on 200 mg/L glyphosate, this concentration seemed suitable to study the expected effect.

## Test

---

The engineered *Bacillus subtilis* carrying goxB under different promoters was incubated on minimal media supplemented with glucose and the targeting herbicide. By optimising UV-vis protocol[3](#ref-3) for glyphosate analysis in minimal media, correlation of measured absorbance to glyphosate concentration was possible [(See results)](https://2022.igem.wiki/lund/results).

## Learn

---

However, practice led to unanticipated outcomes. Glyphosate concentration over time increased. In addition, the expected starting concentration of 200 mg/L in samples without cells could not be verified via this method, leaving strong indications for corrupted derivatization. Through literature research, we knew that *Bacillus* secretes several proteins and peptides especially under environmental changes[4](#ref-4). Amino-groups of these compounds might react with FMOC-Cl, thus contributing to the measured absorbance.

## Re-design

---

In order to detect the level of glyphosate present in the media, these interferences need to be eliminated. By change towards a chromatographic method, not just selectivity but also sensitivity would be increased. Alternatively, using ion exchange chromatography with integrated pulsed amperometric detection would avoid the necessity of upstream derivatisation reactions[5](#ref-5). Other approaches such as LC/MS-MS and LC/FLD have been utilised for glyphosate analysis in environmental samples[6,](#ref-6)[7](#ref-7) and might be a possible application for our project. However, the establishment of a reliable detection method did carry more burdens than expected. The development of one such method should be considered the key point to reach further project progress.

## References

---

1. Van Wachenfeldt, C.

   (2022, July 5). Differences in spore and non-spore forming *Bacillus subtilis*. Personal Communication.
2. Felton, D. E., Ederer, M., Steffens, T., Hartzell, P. L., & Waynant, K. V.

   (2017, November 28). UV–Vis Spectrophotometric Analysis and Quantification of Glyphosate for an Interdisciplinary Undergraduate Laboratory. *Journal of Chemical Education, 95*(1), 136–140.

   [Full text](https://doi.org/10.1021/acs.jchemed.7b00440%20%20%20%20)
3. Rodriguez, J. A., Mendoza-Huizar, L. H., Pérez-Moreno, F., & Carrillo, E. G.

   (2014, March 11). Determination of glyphosate and aminomethlyphosphonic acid in HPLC with Pre-Column derivatization using 1,2-Naphtoquinone-4-sulfonate. *Journal of Liquid Chromatography &Amp Related Technologies, 37*(9), 1298–1309.

   [Full text](https://doi.org/10.1080/10826076.2013.789801%20%20%20%20)
4. Lam, K. H.E., Chow, K. C., Wong, W. K.R.

   (1998). Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins *Journal of Biotechnology, 63*(3), 167-177.

   [Full text](https://pubmed.ncbi.nlm.nih.gov/9803531/%20%20%20%20)
5. Sato, K., Jin, J. Y., Takeuchi, T., Miwa, T., Suenami, K., Takekoshi, Y., & Kanno, S.

   (2001, June). Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography. *Journal of Chromatography A, 919*(2), 313–320.

   [Full text](https://doi.org/10.1016/s0021-9673%2801%2900843-3%20%20%20%20)
6. Kaczyński, P., & Łozowicka, B.

   Liquid chromatographic determination of glyphosate and aminomethylphosphonic acid residues in rapeseed with MS/MS detection or derivatization/fluorescence detection. *Open Chemistry, 13*(1).

   [Full text](https://doi.org/10.1515/chem-2015-0107%20%20%20%20)
7. Hao, C., Morse, D., Morra, F., Zhao, X., Yang, P., & Nunn, B.

   (2011, August). Direct aqueous determination of glyphosate and related compounds by liquid chromatography/tandem mass spectrometry using reversed-phase and weak anion-exchange mixed-mode column. *Journal of Chromatography A, 1218*(33), 5638–5643.

   [Full text](https://doi.org/10.1016/j.chroma.2011.06.070%20%20%20%20)

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{% block title %}Experiments{% endblock %}
{% block lead %}Our wetlab protocols{% endblock %}
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Here, you can find an overview of all the protocols we have used for our experimental part. We would like to thank Johan Bonde and Claes van Wachenfeldt in particular, who were kindly enough to provide us with some of there optimized protocols for cloning in E. coli and transformation into Bacillus subtilis, subsequently.
@ Future iGEMers - Feel free to use these for your own projects. And Good Luck.

---

[Media Formulations](https://static.igem.wiki/teams/4373/wiki/protocols/1-media-formulations.pdf)

[Restriction-based cloning into *E.coli*](https://static.igem.wiki/teams/4373/wiki/protocols/2-restriction-base-cloning-into-ecoli.pdf)

[Gibson Assembly](https://static.igem.wiki/teams/4373/wiki/protocols/3-gibson-assembly.pdf)

[Gel Extraction](https://static.igem.wiki/teams/4373/wiki/protocols/4-gel-extraction.pdf)

[PCR Cleanup](https://static.igem.wiki/teams/4373/wiki/protocols/5-pcr-clean.pdf)

[Plasmid Extraction](https://static.igem.wiki/teams/4373/wiki/protocols/6-plasmid-extraction.pdf)

[Transformation into *E.coli*](https://static.igem.wiki/teams/4373/wiki/protocols/7-transformation-into-ecoli.pdf)

[Transformation into *B.subtilis*](https://static.igem.wiki/teams/4373/wiki/protocols/8-transformation-into-bacillus-subtilis.pdf)

[Starch hydrolysis Test](https://static.igem.wiki/teams/4373/wiki/protocols/9-starch-hydrolysis-test.pdf)

[Protein Expression in *B.subtilis*](https://static.igem.wiki/teams/4373/wiki/protocols/10-protein-expression-in-bacillus-subtilis.pdf)

[SDS PAGE](https://static.igem.wiki/teams/4373/wiki/protocols/11-sds-page.pdf)

[MIC Assay](https://static.igem.wiki/teams/4373/wiki/protocols/12-mic-assay.pdf)

[UV-Vis Spectrophotometry](https://static.igem.wiki/teams/4373/wiki/protocols/13-uv-vis-spec.pdf)

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{% block title %}Results{% endblock %}
{% block lead %}What we achieved in the lab (or didn't){% endblock %}
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# Project Overview

![](https://static.igem.wiki/teams/4373/wiki/result/summary.jpg)

## Part 1 – Cloning

---

### Design of gene construct

With two main pathways identified for glyphosate degradation, the analysis of enzymes involved in the glyphosate reducing step were of highest interest. Sequences encoding for two glyphosate dehydrogenases, *goxA* and *goxB* and one C-P-cleaving multimeric enzyme complex, *phnGHIJK* were obtained from NCBI genebank[1](#ref-1),[2](#ref-2),[3](#ref-3). Codons were modulated for expression in *Bacillus subtilis* using IDT codon optimizer tool.

Regulation of high-level protein production within the bee gut microbiome needs to be considered for the choice of promoter. Here, the strong constitutive promoter Pveg would provide constant protein expression during all growth phases. However, continuous expression may lead to increase in stress-level for the cells, inhibiting the recombinant protein production process. To enable better control over this process, a construct under IPTG-inducible promoter Physp is introduced. A constant feed of inducer would be required for application in *Apis mellifera* making it not feasible as a probiotic. To test the proof of concept, it’s effects will still be analysed. Furthermore, a third promoter was acquired. The strong stationary phase promoter, Pylb exhibits protein expression when late log phase/early stationary phase is reached[4](#ref-4). Hereby, glyphosate degrading proteins are only produced upon sufficient growth meaning colonisation of bees gut microbiome. When come into contact with other non-target organisms, this regulatory response creates an additional safety mechanism.

Besides different promoter regions, each gene of interest (GOI) was flanked upstream with an 5’UTR-region including a *Bacillus subtilis* specific ribosome binding site (RBS) as well as two stop codons and a terminator sequence downstream of the gene. For restriction-based cloning, 5’-HindIII and 3’-SalI (for pCW7) and NheI (for pCW101) restriction sites were introduced. The inserts as seen in Figure 1 were synthesised by IDT and TWIST Bioscience.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-1.png)

### Design of vector

Two strategies for recombinant protein production were followed – chromosomal integration and plasmid expression. Specific functional units are considered in the design of the vector plasmid. The low copy-number plasmid pCW7 carries two *ori* allowing replication within *E. coli* and *B. subtilis* and thus suitable as a shuttle vector. However, homologous recombination requires a common set of integration sites present in chromosomal DNA and plasmid. Here, pCW101 inhabits *amyE* integration sites used for chromosomal integration in *B. subtilis.*
Both vectors were kindly provided by Claes von Wachenfeldt.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-2.png)

### Restriction and Ligation

Traditional cloning was the method of choice for the introduction of our GOI into the designed vector. Therefore, digestion of the synthesised GOI carrying specific restriction sites as well as the vector was performed. Cloning into pCW7 was based on creating 5’- and 3’-end sticky ends by using HindIII and SalI, respectively. A different 3’-end was created for cloning into pCW101. Although carrying the same restriction site, their close proximity was assumed to create improper overhangs thus reducing ligation specificity. NheI was therefore used for cloning work into pCW101 backbones. Ligation reaction was performed with T4 ligase ([See further protocols](https://2022.igem.wiki/lund/notebook)).

With the described method, transformants for *goxB* inhabiting the ylb and hysp promoter inside the integrative pCW101 plasmid were obtained. To evaluate these constructs, colony PCR with backbone specific primers should allow amplification of integrated fragments. Figure 3 A shows clear distinguishable bands for several clones for pCW101-*goxB*-ylb and pCW101-*goxB*-hysp at around 1600 bp (size of fragment + 260 bp generated by primers binding inside the vector backbone). Sequence similarity of these transformants was further tested by sequencing. The constructs analysed showed 100% alignment (Data not shown) – providing proof for successful cloning.

However, no transformants could be obtained with traditional cloning for *goxA* or *phnGHIJK* fragments. Further, ligation into the pCW7 backbone did not produce any constructs whatsoever. Shifting to the exonuclease based cloning method called Gibson Assembly® was expected to increase cloning efficiency ([See Engineering success](https://2022.igem.wiki/lund/engineering)) . Here, transformants of correctly assembled pCW101-*goxB*-veg as well as one pCW7-*goxB*-ylb fragment could be obtained (Figure 3 B&C). Sequencing results concluded homologous sequence similarity for pCW101-*goxB*-veg (Data not shown). However, sequencing of pCW7-*goxB*-ylb disclosed many missense mutations to be present inside the gene and was therefore not considered for further analysis.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-3-2.png)

**Figure 3|** Agarose images of Colony PCR performed in *E. coli* transformed with *goxB-*constructs. (A)pCW101-*goxB*ylb and pCW101-*goxB*hysp (D3). Both fragments were cloned via restriction and ligation using 5'-HindIII and 3'SalI as restriciton sites. (B) *goxB*-veg cloned into pCW101 via Gibson Assembly. (C) *goxB*-ylb cloned into pCW101 via Gibson Assembly.

![](https://static.igem.wiki/teams/4373/wiki/result/pic-1.png)

## Part 2 - Chromosomal integration into Bacillus subtilis

---

![](https://static.igem.wiki/teams/4373/wiki/result/homologous-recombiantion.jpg)
For our designed probiotic, *Bacillus subtilis* was chosen as the optimal expression host. Transformation of the constructed plasmids into our target organisms was performed on the basis of self-induced competence. The generated constructs replace α-amylase encoding genes inside the *Bacillus* chromosome via homologous recombination. Upon integration, the strain loses the ability to degrade starch, a characteristic that can be utilised to determine correct chromosomal incorporation.

This analysis method was used to determine successful *goxB* transformants. Figure 4A shows cells of non-transformed *Bacillus*. Areas surrounding these colonies appear clear implying starch hydrolysis. Within the second half of starch agar plates seen in Figure 4 B-C, the dark coloured medium indicates the presence of bacteria with correctly integrated fragments. Colonies growing on the upper part however show similar behaviour as non-transformed *Bacillus*. It is assumed that these cells either failed to integrate our GOI or low transformation efficiency resulted in low to no uptake of recombinant plasmid.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-4.png)

![](https://static.igem.wiki/teams/4373/wiki/result/pic-2.png)

### Protein Expression

![](https://static.igem.wiki/teams/4373/wiki/result/fig-5-1.png)
To develop an active glyphosate degradation system, overexpression of the goxB encoding gene is necessary. Therefore, overall protein production and thus the functionality of the introduced promoter was assessed via SDS. Cultivation of recombinant *Bacillus* was hereby performed in minimal media supplemented with glucose and glyphosate. To simulate growth conditions similar to the gut microbiome of bees, cells were incubated at 35°C – the average temperature of bees body temperature within the beehive[5](%E2%80%9D#ref_5”).

Samples at different time points were taken and analysed via SDS. Figure 5 shows two distinguishable bands with sizes around 20 and 55 kDa visible in all lanes. Between 40 and 50 kDa, smeared areas indicate the presence of further proteins.

Overexpression of glyphosate dehydrogenase under different promoters is expected to result in high intense bands with a molecular weight of 46 kDa. However, neither a band is visible in this area nor a distinction between WT and *goxB* carrying strains can be made.

To eliminate the possibility of deviant separation of either the marker or protein sample, bands at 20 and 55 kDa were analysed via mass spectrometry and screened for potential proteins within the NCBI database. Ladder was identified as vegetative catalase, an extracellular enzyme, secreted upon oxidative stress[6](#ref-6). Furthermore the second protein can be connected to alkyl hydroperoxide reductase C – another enzyme playing a crucial role in the protection of cells against oxidative stress[7](#ref-7). Additionally the smeared area between 40 and 50 kDa was inspected for evidence of target protein production. However, no peptides responding to glyphosate dehydrogenase could be identified.

Moreover, samples of the supernatant after 24 h cultivation were taken and further studied for proteins secreted into the media. Here, only bands corresponding to the oxidative stress reducing proteins were recognised (Data not shown). However, this does not exclude potential target protein secretion as the supernatant was highly diluted and thereby strongly reduced the intensity of a possible low expressed goxB.

![](https://static.igem.wiki/teams/4373/wiki/result/pic-3.png)

## Part 3 – Glyphosate Degradation

---

### Minimal Inhibitory assay

Glyphosate strongly inhibits bacterial growth. To determine, the lowest concentration on which 100 % of growth is inhibited, an minimal inhibitory concentration (MIC) assay was performed. Here, *B. subtilis* 1S129 WT next to engineered strains carrying the goxB encoding gene were analysed. Cells from overnight cultures grown in minimal media supplemented with glucose and antibiotics (OD450~0.5-07) were plated on minimal media plates with RoundUp©. RoundUp© is a commonly used herbicide made of concentrated glyphosate (360 g/L) dissolved in potassium salt (411 g/L). This chemical was supplemented in different concentration to agar plates (50 mg/L to 4 g/L). Additionally, IPTG was further added to strains with goxB under IPTG-inducible Physp.

**Table 1**| Minimal inhibitor concentration (MIC) of engineered *B. subtilis* 1S129 with *goxB* on different concentrations of RoundupTm (Glyphosate 360 g/L). Cells were incubated at 35°C for 5 days.n=2

|  | WT | PCW101 | goxB-veg | goxB-ylb | goxB-hysp |
| --- | --- | --- | --- | --- | --- |
| MIC [g/L] | 0.5 | 0.5 | 1 | 0.5 | 0.5 |

For a time period of 5 days, plates were inspected for growth. After the first day, colonies on plates with up to 200 mg/L glyphosate concentrations were detected. Growth was hereby visible for all tested strains. However, further incubation resulted in enhanced growth for *B. subtilis* strain carrying goxB under veg promoter up to 1 g/L. All other variants were inhibited by this glyphosate concentration. In comparison to the WT, only goxB-veg produced an altering MIC thus indicating that the introduced glyphosate dehydrogenase contributes to greater cell survival. Based on these results *B. subtilis* 1S129 seem to generally carry a high tolerance against glyphosate, a highly beneficial characteristic for usage as probiotic in bees. Limited glyphosate resistance has further been reported for some *B. subtilis* species[8](#ref-8). Nevertheless, such high tolerance was reported for the first time in this strain.

![](https://static.igem.wiki/teams/4373/wiki/result/pic-4.png)

### Growth experiment on glyphosate

Regular OD measurements of cells incubated with glyphosate should give insight about the individual growth behaviour of each variant. For the selection of glyphosate concentration 200 mg/L were used - the MIC for *B. subtilis* after 24 h. After inoculation of cultivation media, cultures were incubated for 2 hours to reach the start of log-phase. Glyphosate in the form of RoundUp© was then added. Note that for variant goxB-hysp, IPTG was added first and incubated for another 2 hours to induce protein expression before glyphosate supplementation.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-6.png)

Difference in growth between strains w/o and w/ glyphosate were expected. However, from Figure 6, a clear distinction between each growth curve is difficult. Furthermore, *B. subtilis* WT expresses slightly enhanced growth compared to organisms with integrated goxB coding genes. Expression of these proteins may increase the cell's stress level therefore slowing down its growth. As no difference between growth on glyphosate compared to no glyphosate cultivation is visible, the cells metabolism seem not to be affected by addition of this chemical. The slight alteration in regard to the WT strain can be reduced to the metabolic burden generated by recombinant protein expression. However, this hypothesis could not be supported by generated SDS results (see Figure 5)

![](https://static.igem.wiki/teams/4373/wiki/result/pic-5.png)

### Observation of glyphosate degradation using UV-vis

As described above, glyphosate does not seem to impact growth of the engineered bacteria – making it a great probiotic host. To further study the degradation efficiency of the introduced glyphosate dehydrogenase, samples of culture supplemented with glyphosate (RoundUP©) were taken every 24 h. Cultivation was performed under the same conditions as defined above. External glyphosate concentrations were measured via UV-vis spectrophotometry[9](#ref-9).

In order to obtain absorbance, glyphosate had to undergo an additional derivatisation process based on FMOC-Cl. This light absorbing compound reacts with glyphosates’ amino-group making glyphosate detectable.

For correlation of measured absorbance to glyphosate concentration, a standard curve was created (Figure 7). From this linear regression, the glyphosate concentration in the supernatant could be determined. Expressed glyphosate dehydrogenase should break down assimilated glyphosate, thus reducing the external glyphosate concentration inside the media. With a starting value of 200 mg/L, a steady decline over time was expected. However, Figure 8 shows the contrary. Even firstly observed reductions after 24 h cannot explain the elevated level of glyphosate above the threshold of 200 mg/L after 48 h – in all samples. Furthermore, to exclude general degradation of glyphosate at cultivation conditions, a control of glyphosate in minimal media without cells was analysed. Hereby, no glyphosate could be detected within these samples (Data not shown). This strongly indicates that the measured absorbance did not represent the actual glyphosate concentration.

![](https://static.igem.wiki/teams/4373/wiki/result/fig-7-8.png)

![](https://static.igem.wiki/teams/4373/wiki/result/fig-9.png)

In order to make sense of these results, one has to look back at the derivatisation reaction (Figure 9). The utilized derivatisation agent FMOC-Cl binds to the amino group – a functional group not only present in glyphosate. With peptides and proteins consisting of amino acids carrying this functional group, interaction with secreted proteins is very likely. Hereby, *Bacillus subtilis* in particular is known for its many secretory pathways. Furthermore, Yamane et al. described a strong connection between protein secretion and environmental changes[10](#ref-10). By cultivation on glyphosate, this effect might be induced. The increase in absorbance would thus correlate towards growth related peptide or protein secretion. However, this does not explain the low measured absorbance in samples incubated with only glyphosate and media. One explanation might lay in different stock solutions employed for standard curve and degradation analysis. For the standard curve highly pure analytical glyphosate was used. All degradation experiments however were performed with RoundUp©. The concentration of glyphosate (360 g/L) in this product was not further confirmed and relied only on the package description. Alterations in the starting glyphosate content would have further influenced the amount within the culture, giving non reliable results.

![](https://static.igem.wiki/teams/4373/wiki/result/pic-6.png)

## Discussion and conclusion

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The focus of this project rested in the construction of different glyphosate degrading systems in non-spore forming *Bacillus subtilis.* Three enzymes under the control of different promoter functions should be analysed and compared in terms of degradation efficiency under conditions similar to bees gut microbiome. Therefore, integration of these protein encoding genes into Bacillus chromosomal DNA was the main approach. Here, we successfully cloned *goxB* into an integrative vector and transformed it into *E. coli* to achieve high plasmid replication*.* Transformation of these obtained recombinant plasmids into *Bacillus* generated three engineered strains with goxB + promoter integrated into their chromosomal DNA. However, subsequent expressions did not produce the corresponding protein. By changing the detection method from SDS to western blot, the presence of our target protein might be defined when using highly sensitive antibodies. Nevertheless, protein expression might be generally suppressed. The *lac* operon is still present inside the integrated fragment even with new introduced promoters. Potential *lac* repressor binding sites within the promoter regions of Pveg and Pylb might contribute to inhibited protein expression. Nevertheless, the construct carrying Physp should still produce our target protein. Thus, sequencing of the integrated genes for mutations inside the promoter might provide more insight on why the system failed to produce any proteins that could be detected.

Even though protein expression could not be directly confirmed, analysis of degradation activity through the introduced glyphosate dehydrogenase may provide evidence for its production. Several methods should screen for differences in behaviour of the engineered bacteria. MIC assay provided evidence for increased survival within goxB-veg carrying *Bacillus* at higher glyphosate concentrations. However, studying glyphosate related effects on growth did not give a clear distinction between native and altered strains. Furthermore, activity evaluation on glyphosate based on spectrophotometric measurements did not produce any reliable results. Glyphosate unspecific binding of the derivatization agent FMOC-Cl can be considered as the methods bottle-neck. Highly sensitive chromatographic methods such as LC/MS-MS might generate more selective results[6](#ref-11). However, by relying on derivatization agents binding to the amino group, undesirable binding to secreted proteins and peptides will always accompany these outcomes. Thus, a reliable detection method needs to be established to determine glyphosate breakdown of cell-based systems.

Nevertheless, from enhanced MIC for *goxB*-veg integrated in *B. subtilis* alone, **no proof** of a functioning glyphosate degradation system can be concluded.

## References

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   (2022, October 5).Uncultured bacterium clone pGOXA FAD-dependent glyphosate oxidase. *NCBI Genebank*.

   [Full text](https://www.ncbi.nlm.nih.gov/nucleotide/GU479462.1?report=genbank&log$=nucltop&blast_rank=2&RID=6XG6GV75016)
2. NCBI-genebank

   (2022, October 5).Uncultured bacterium clone pGOXB FAD-dependent glyphosate oxidase. *NCBI Genebank*.

   [Full text](https://www.ncbi.nlm.nih.gov/nucleotide/GU479463.1?report=genbank&log$=nucltop&blast_rank=5&RID=6XG6GV75016)
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