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). 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).
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 strains1. 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 nm2.
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 protocol3 for glyphosate analysis in minimal media, correlation of measured absorbance to glyphosate concentration was possible (See 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 changes4. 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 reactions5. Other approaches such as LC/MS-MS and LC/FLD have been utilised for glyphosate analysis in environmental samples6,7 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
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(2022, July 5). Differences in spore and non-spore forming Bacillus subtilis. Personal Communication.
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(2017, November 28). UV–Vis Spectrophotometric Analysis and Quantification of Glyphosate for an Interdisciplinary Undergraduate Laboratory. Journal of Chemical Education, 95(1), 136–140.
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(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.
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(1998). Construction of an efficient Bacillus subtilis system for extracellular production of heterologous proteins Journal of Biotechnology, 63(3), 167-177.
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(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.
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Liquid chromatographic determination of glyphosate and aminomethylphosphonic acid residues in rapeseed with MS/MS detection or derivatization/fluorescence detection. Open Chemistry, 13(1).
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(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.