Team:Nottingham/Results


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Results



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Project
Results

Preliminary Characterisation of C. sporogenes

The growth profile of Clostridium sporogenes was determined in TYG media over 48 hours. Through the measurement of the pH change, Optical Density at 600 nm (OD600) and acetate production, it was found that with the exception of the pH measurements, all of the variables followed our predictions, as demonstrated in Figures 1 and 2. In Figure 1, it can be seen that C. sporogenes has an initial lag phase of 3 hours, at which point the cultures progress into exponential growth for 7 hours, the cultures then enter stationary phase at 10 hours and finally death phase when the OD600 of the culture declines.


Figure 1. The Optical Density (OD) of wildtype C. sporogenes. OD600 measurements were taken every hour from 0h to 8h, and at 24h and 48h

Figure 2. The product profile of wildtype C. sporogenes during growth in TYG media. 1 ml samples of the supernatant were taken for gas chromatography (GC) analysis at 2, 4, 6, 8, 24 and 48 hours.

In Figure 2, the concentration of acetate is continuously increasing as measured by both Gas Chromatography (GC). Acetate is the precursor to acetone, therefore an accumulation of acetate indicates that a mutant of C. sporogenes harbouring the acetone production operon, should have the capacity to produce acetone. Both analyses also show the production of approximately 10 mM of ethanol, however this is not a risk factor to the project ethanol is not toxic to C. sporogenes at these concentrations.[1]


Acetate is acidic; therefore, an accumulation of acetate is predicted to cause a drop in pH. This is a potential problem to the project as the acetoacetate decarboxylase (Adc) enzyme, necessary to catalyse acetone production from acetate, is pH sensitive; Adc has an optimum pH of 5.5 but can work effectively up to approximately a pH of 6.5.[2] As shown in Figure 3, the pH of the media reaches levels in excess of 7.5 at 48 hours, clearly exceeding the limit of the Adc enzyme’s optimal pH range, and therefore further experimentation must be conducted to determine the optimum growth medium for our mutant C. sporogenes strains.


Figure 3. The pH of wildtype C. sporogenes over a period of 48 hours, grown in TYG media. pH was measured every hour from 0h to 8h, and at 24h and 48h.

Figures 4a and 4b show that there is little difference between the concentration of acetate and ethanol detected by use of either method of High-Performance Liquid Chromatography (HPLC) or GC. Though the GC is able to detect a wider range of chemicals, the HPLC is just as useful to the project as it is able to detect acetate. Therefore, in future experiments either GC or HPLC was used, depending on their availability within the lab.


Figure 4a. The concentration of Ethanol produced by wildtype C. sporogenes. 1 ml samples of the supernatant were taken for GC and High-Performance Liquid Chromatography (HPLC) analysis at 2, 4, 6, 8, 24 and 48 hours.

Figure 4b. The concentration of Acetate produced by wildtype C. sporogenes. 1 ml samples of the supernatant were taken for GC and High-Performance Liquid Chromatography (HPLC) analysis at 2, 4, 6, 8, 24 and 48 hours.

Problem: pH range not appropriate for Adc to work


To investigate this problem further, an experiment was designed to determine the best growth media for C. sporogenes which would reduce the pH to within the Adc enzyme working range and support the growth of the organism. The wild type C. sporogenes strain was grown in a range of glucose concentrations in TYG media:

  • TYG corrected to pH 5.5 with HCL (aq)
  • TYG + 30 mM glucose
  • TYG + 60 mM glucose
  • TYG + 100 mM glucose

The data from the lab indicated that the addition of glucose would increase pyruvic acid production, thus optimising growth and reducing the pH of the media, allowing the Adc enzyme to function efficiently. Over 48 hours the OD600 and pH of the media were measured and samples were taken for GC analysis, as illustrated in Figures 5, 6 and 7.


Figure 6 shows that all the glucose concentrations can be used to produce acetone, as all the pHs reached or were below the maximum threshold of pH 6.5, with 100 mM of glucose exhibiting the largest reduction in pH. However, as shown in Figure 5, the 100 mM glucose supplemented media causes attenuation in growth of C. sporogenes, whilst the most optimal media for growth is TYG supplemented with 30 mM of glucose. Furthermore, Figure 7 shows that media supplemented with 30 mM glucose results in the highest concentration of acetate, the precursor to acetone, from 8 hours onwards.


As observed, TYG media supplemented with 30 mM glucose reaches a maximum pH of 6.7 at 48 hours, whilst resulting in the highest concentration of acetate produced and the least attenuation of C. sporogenes growth. We therefore concluded that the best media for future experiments to be conducted in is TYG supplemented with 30 mM glucose.


Figure 5. Optical Density (OD) of wildtype C. sporogenes grown in TYG media with increasing concentrations of glucose. OD600 was measured of C. sporogenes grown in TYG containing, 30 mM, 60 mM and 100 mM of glucose, and TYG media corrected to pH 5.5 using HCl. Measurements were taken every hour from 0h to 8h, and at 24h and 48h

Figure 6. pH over time of wildtype C. sporogenes grown in TYG media with increasing concentrations of glucose. The pH of C. sporogenes was measured in TYG containing 30 mM, 60 mM and 100 mM of glucose, and TYG media corrected to pH 5.5 with HCl. pH was measured every hour from 0h to 8h, and at 24h and 48h.

Figure 7. Concentration of Acetate produced by wildtype C. sporogenes grown in TYG media with increasing concentrations of glucose. Supernatant samples were taken for High-Performance Liquid Chromatography (HPLC) analysis at 0, 4, 8, 24 and 48 hours.

Does acetone kill C. sporogenes?


An experiment was conducted to determine what concentration threshold of acetone could be tolerated by C. sporogenes cells. The acetone producing organism Clostridium acetobutylicum was used as a guide for the concentration of acetone that could potentially be produced by C. sporogenes, which is approximately 70 mM.[3] In order to simulate the conditions of high acetone yields, varying concentrations of acetone in TYG were set up: 0 mM, 100 mM, 200 mM, 300 mM and 400 mM. Over 48 hours, OD600 and Colony Forming Units (CFU) were measured to determine the growth profile of C. sporogenes under these conditions.


Figures 8 and 9 indicate that at acetone concentrations up to 400 mM, the cells remain viable. On further consultation with an expert on clostridial growth in acetone conditions, it was predicted that acetone would not attenuate the growth of C. sporogenes, up to a concentration of 0.5 M. However, it is unlikely that the mutant can produce these quantities of acetone, so this should not prove to be a factor in our experiments. This validated the viability of acetone as a reporter due to it being; non-toxic, easily detectable and a volatile substance.



Figure 8. The Optical Density (OD) of wildtype C. sporogenes grown in TYG media with increasing concentrations of acetone. OD600 was measured of C. sporogenes grown in TYG with 30 mM glucose containing  0 mM, 100 mM, 200 mM, 300 mM and 400 mM of acetone. Measurements were taken at 24- and 48-hour intervals.

Figure 9. Colony Forming Units per ml (CFU/ml) of wildtype C. sporogenes grown in TYG media with increasing concentrations of acetone. CFU/mL was measured of C. sporogenes grown in TYG with 30 mM glucose containing 0 mM, 100 mM, 200 mM, 300 mM and 400 mM of acetone. Measurements were taken at 24- and 48-hour intervals.

Carrot Juice Experiments


Based on the advice of Professor Mike Peck, an experiment was conducted to determine whether carrot juice could support the growth of C. sporogenes. Prof. Peck referred to outbreaks of botulism which occurred in Florida and Georgia in 2006, due to the inappropriate storage of carrot juice.[4] The aim of the experiment was to determine whether carrot juice could also support the growth of C. sporogenes, as a proof that it can grow in real food media.


Varying dilutions of carrot juice were made from fresh carrots: 10%, 30%, 50% and 100% in phosphate buffered saline (PBS). We used fresh carrots as commercially available carrot juice is pH corrected to be more acidic and less hospitable to foodborne pathogens. This practice is as a direct result of the botulism outbreaks forcing changes on the industry to prevent further outbreaks. Over 48 hours, CFU were measured to determine the growth characteristics of C. sporogenes in the carrot juice, and samples for GC analysis were also taken to demonstrate the production of acetate in the carrot juice media.


As can be seen in Figure 10, the 50 % and 100 % carrot juice media supported the highest C. sporogenes growth, with 24 hour average CFU measurements of 1.5 x 107 and 4.1 x 107 respectively, three orders of magnitude higher than the average CFU count for 30 % and four orders of magnitude higher than 10 % carrot juice, with 2.2 x 103 and 4.4 x 102 respectively. This indicates that C. sporogenes can grow in real food media, confirming C. sporogenes as a good choice of surrogate to C. botulinum.

Figure 10. Colony Forming Units per ml (CFU/ml) of wildtype C. sporogenes grown in increasing concentrations of carrot juice. C. sporogenes was grown in carrot juice at concentrations of 10%, 30%, 50% and 100%. CFU/ml was measured at 24 hours for each concentration.

Integrating botR-Expression Modules into the pyrE Locus C. sporogenes Genome

Construction of the botR-Expression Modules


Three botR-expression modules comprising of the botR sigma factor gene under the control of no promoter, the native PbotR promoter and the inducible PLAC system were assembled by linear ligation. These modules were then inserted by ligation in to the RibosCas CRISPR/Cas9 vector backbone, pRE-Cas1-p15a[5], and stored in the E. coli cloning host TOP10. Two RiboCas vectors were constructed, with two separate sgRNA sequences to direct the Cas9 mediated insertion of the modules into the pyrE locus of the C. sporogenes genome.


Plasmid specific flanking primers were used to verify the insertion of the expression modules by colony PCR (Figure 11). The assembled plasmids were further verified by Sanger sequencing and subsequently transformed into an E. coli conjugal donor variant of the TOP10 strain, designated TOPSEX.


Figure 11. Gel electrophoresis image of flanking PCR for insertion of the three botR-expression modules in to pRE-Cas1-p15a, for sgRNA 1 (A) and 2 (B). The PpyrKDE insert has a band with a size of 2224 bp, Pntnh has a band size of 2436 bp and PLAC has a band size of 3607 bp.

Integration of botR-Expression Modules into the Genome of C. sporogenes by CRISPR/Cas9


The RiboCas plasmids for the integration of the three botR-expression modules were transferred in to C. sporogenes by conjugation. Transconjugant colonies were restreaked on to TYG plates supplemented with 5 mM theophylline to induce the expression Cas9 induction. Putative integrant colonies were screened with external chromosomal and internal module primers to confirm integration (Figure 12 A), and flanking primers for sequencing (Figure 12 B). Integrant mutants were also subjected to plasmid loss assays and confirmed by PCR, using plasmid specific primers (Figure 12 C). Integrant mutants were obtained for both sgRNA 1 and 2 RiboCas plasmids.


Figure 12. A. PCR screen to confirm integration of the three botR expression modules into the genome at the pyrE locus; B: PCR screen with flanking primers to amplify the entire botR-expression modules for sequencing; C: PCR screen to confirm the loss of the RiboCas plasmid from integrant mutants.

Generation of Reporter Expressing Strains

Construction of the Reporter Plasmids


Four reporter plasmids were constructed; C. acetobutylicum APO, C. botulinum APO, gusA and FAST. Each of these reporters was cloned downstream of Pntnh, Pfdx as a positive control and with no promoter as a negative control. The reporter plasmids were assembled by HiFi assembly, using the vector backbone pMTL82151, with the exception of the gusA reporter being assembled in to pMTL82121, which harbours a low copy Gram-negative replicon. Assembled vectors were transformed in to the E. coli TOP10 cloning host and subsequently the conjugal donor variant, TOPSEX for transfer in to the botR expressing C. sporogenes host strains.

Conjugating the Reporter Plasmids into the botR-Expressing C. sporogenes


The reporter plasmids were transferred by conjugation in to the botR expressing C. sporogenes hosts; PpyrKDE, PbotR, PLAC and the botR negative wild type strain. Transconjugant colonies were restreaked to purity and PCR screened with plasmid specific flanking primers to confirm the presence of the reporter vectors and for sequence verification (Figure 13).


Table 1: Summary of Host Strains and Conjugated Plasmids


Host

Conjugated Plasmids

C. sporogenes ΔpyrE::PpyrKDE-botR

pMTL82151-Pntnh-FAST
 

pMTL82121-Pntnh-gusA
 

pMTL82151-Pntnh-APO
 

pMTL82151-Pntnh-APO-Cb

C. sporogenes ΔpyrE::PbotR-botR

pMTL82151-Pntnh-FAST
 

pMTL82121-Pntnh-gusA
 

pMTL82151-Pntnh-APO
 

pMTL82151-Pntnh-APO-Cb

C. sporogenes ΔpyrE::PLAC-botR

pMTL82151-Pntnh-FAST
 

pMTL82121-Pntnh-gusA
 

pMTL82151-Pntnh-APO
 

pMTL82151-Pntnh-APO-Cb

C. sporogenes wildtype

pMTL82151-Pntnh-FAST
 

pMTL82151-Pfdx-FAST
 

pMTL82151-FAST
 

pMTL82121-Pntnh-gusA
 

pMTL82121-Pfdx-gusA
 

pMTL82121-gusA
 

pMTL82151-Pntnh-APO
 

pMTL82151-Pfdx-APO
 

pMTL82151-APO
 

pMTL82151-Pntnh-APO-Cb
 

pMTL82151-APO-Cb

Figure 13. Generation of Reporter-Expressing Strains. Colony PCR on biological replicates (1-3) of strains harbouring; A: C. acetobutylicum APO B: GusA, C: FAST and D: C. botulinum APO reporter-expressing plasmids. The host strain, promoter and reporter combinations are indicated.

Assaying Reporter Expression

Reporter characterisation assays were performed over a period of 48 hours with OD600 measurements taken at 0, 4, 8, 24 and 48 hours and samples taken at 8, 24 and 48 hours to assay the respective reporters.


We measured the level of fluorescence in the gusA and FAST reporters using the Clairostar plate reader and the level of acetone (in the two APO constructs) using gas chromatography.


Table 2: Summary of Strain and Reporter Combinations

Strain/Reporter Designation

Genotype

Description

PpyrKDE

C. sporogenesΔpyrE::PpyrKDE-botR + pMTL82151/pMTL82121-Pntnh-reporter

Engineered strain with botR sigma factor expression driven by the PpyrKDE promoter, harbouring the reporter vector, under the control of the BotR-dependent Pntnh promoter

PbotR

C. sporogenesΔpyrE::PbotR-botR + pMTL82151/pMTL82121-Pntnh-reporter

Engineered strain with botRsigma factor expression driven by the PbotRpromoter, harbouring the reporter vector, under the control of the BotR-dependent Pntnh promoter

No botR

C. sporogenes wildtype + pMTL82151/pMTL82121-Pntnh-reporter

Wildtype strain harbouring the reporter vector, under control of the BotR-dependent Pntnh promoter.

Pfdx (+ve control)

C. sporogenes wildtype + pMTL82151/pMTL82121-Pfdx-reporter

Positive control; Wildtype strain, harbouring the reporter vector, under the control of the constitutive native Pfdx promoter.

-ve control

C. sporogenes wildtype + pMTL82151/pMTL82121-No promoter-reporter

Negative control; Wildtype strain, harbouring the reporter vector with no promoter.

Acetone

GusA

FAST

Acetone Reporters: C. botulinum and C. acetobutylicum Constructs


Figure 14a shows acetone production for the engineered C. sporogenes host strains harbouring the C. acetobutylicum APO plasmids.


The PbotR reporter strain shows a steady increase in acetone production from 8 hours, with production peaking at 48 hours, with an average acetone concentration of 2.7 mM.


The positive control, with the constitutive Pfdx promotor, exhibits significantly higher acetone production than any of the other constructs, initial acetone production begins higher than all other constructs at 1.25 mM, peaking at 48 hours with an average reading of 5.3 mM.


The PpyrKDE strain functions as an additional positive control, with the botR gene under constitutive expression of the PpyrKDE ­at the pyrE locus. This strain shows a lower yield in acetone production than the PbotR strain, also peaking at 48 hours at an average concentration of 1.25 mM.


The negative control and no botR strains, as expected, have low-levels of acetone production, with concentrations of 0.2 and 0.5 mM respectively.


Figure 14b shows the OD600 of constructs increasing exponentially between 0 and 8 hours. However, this is where the growth profile of the strain harbouring the construct with the APO under the control of the Pfdx promoter and the rest of the constructs diverge. The Pfdx promoter OD600 peaks at a lower point but maintains a slight increase in OD until 24 hours, where OD drops to the same level as the other constructs. The rest of the constructs follow the same pattern, of growth of peaking at 8 hours, and decreasing by roughly a third at 24 hours and then slowly declining in OD600 to 48 hours. The different growth pattern exhibited by the Pfdx construct can be attributed to the fact that Pfdx is a native C. spororgenes promoter which is active earlier during exponential phase of growth, as can be seen in the acetone production data, placing a greater metabolic burden on the cells and attenuating growth.


The obtained yields have mean values of approximately 2.6 mM and 5.3 mM represent concentrations that could be detectable by our electronic nose. These results validated our modelling predictions that addition of specific genes (namely ctfA/B and adc) would provide the pathway required for C. sporogenes to make acetone from acetate, provided that the strain is cultured in media with a suitable pH range.

Figure 14a. Non-normalised acetone production of the engineered C. sporogenes strains harbouring the APO reporter constructs, over 48 hours. Three biological replicates of the strain were grown in TYG supplemented with 30 mM glucose, with 1 ml samples taken at 8, 24 and 48 hour timepoints for GC analysis.

Figure 14b. Optical Density at 600 nm (OD600) of all the engineered C. sporogenes harbouring the APO reporter constructs, over 48 hours. OD600 was measured for all constructs grown in TYG supplemented with 30 mM glucose, measurements were taken at 0, 4, 8, 24 and 48 hours. 

Characterisation of Reporter Strains vs Wild Type

The growth and sporulation profiles of the engineered reporter strains (ΔpyrE::PbotR-botR + pMTL82151/pMTL82121-Pntnh-reporter) were determined. This was to confirm that important properties of the wildtype strain had not been compromised by the engineering activities undertaken, or by the expression of the selected reporters. OD600 was measured over a period of 48 hrs while the sporulation capacity was assayed at 120 hr, expecting that at that timepoint the entire cell population would be in spore form.


Figure 21 shows all reporter strains grow at a relatively similar rate to the wildtype strain, with only slight deviations visible after the stationary phase. Likewise, as seen in Figure 22, there are no significant differences between the total (non heat-treated) and sporulating (heat-treated) colony forming units for all reporter strains, suggesting that their sporulating and germinating capacity has not been affected.


Overall it can be concluded that the important phenotypic characteristics of the parent strain (C. sporogenes) have not been compromised by the introduction of our reporter expression circuit, confirming its suitability for the creation of our Notox technology.


Figure 21. Optical density at 600 nm (OD600) of the engineered reporter strains alongside the wildtype strain (WT) of C. sporogenes, over 48 hours. OD600 was measured of all reporter strains alongside the wildtype strain. OD600 was measured for all constructs grown in TYG supplemented with 30 mM glucose, measurements were taken at 0, 2, 4, 8, 10, 24 and 48 hours. Data represent mean values of three independent cultures for WT and of three biological replicates for all engineered strains ± SD.

Figure 22. Sporulation/germination capacity of engineered reporter strains alongside the wildtype strain (WT) of C. sporogenes, at 120 hours of incubation; sporulating CFUs correspond to the number of spores with germinating ability after heat-treatment and total CFUs correspond to the total cell count before heat-treatment. Data represent mean values of three independent cultures for WT and of three biological replicates for all engineered strains ± SD. Statistical analysis was carried out using two-tailed unpaired t-test; p-values are indicated as: 0.1234 (ns), 0.0332 (*), 0.0002 (***), <0.0001 (****).

Comparison ctfA/B from C. acetobutylicum vs C. botulinum

The ctfA/B & adc genes enable the production of acetone from acetate. Both ctfA and ctfB are present within C. acetobutylicum and C. botulinum, however not in C. sporogenes. Unlike C. acetobutylicum, acetone production within C. botulinum (and C. sporogenes) is not possible due to a lack of the adc gene. The rest of the genes in the ABE fermentation pathway are present within C. botulinum and C. sporogenes. Therefore, under the assumption that the missing acetone-production genes could be added to C. sporogenes (or C. botulinum), a model was created to predict whether C. sporogenes could have a complete and possibly favourable acetone-production pathway.


The ctfA/B genes from C. acetobutylicum and C. botulinum were compared to establish which would give the most favourable acetone yield within C. sporogenes. For any future work with this reporter concept on C. botulinum (replacing the neurotoxin with an acetone reporter) there needed to be conclusions on whether the native ctfA/B genes are able to produce adequate levels of acetone for precise analysis of what would be neurotoxin production. As C. botulinum is currently unable to produce acetone, it is uncertain if the ctfA/B genes are functional, and to what degree.


The data in Figure 23 indicates C. acetobutylicum ctfA/B genes have a much greater efficacy and therefore enable significantly higher acetone production. This can be seen from the 10-fold increase in acetone production when ctfA/B from C. acetobutylicum is used compared to when ctfA/B from C. botulinum is used in the acetone production operon.


From this data, it is recommended that’s further use of this acetone reporter concept for future C. botulinum studies use ctfA/B genes from C. acetobutylicum. Native ctfA/B genes may also need deleting from C. botulinum to create the final reporter strain, as they may produce background acetone readings which would affect the sensitivity and accuracy of the results.


Figure 23. Comparison of acetone production in the C. sporogenes ΔpyrE::PbotR-botR strain harbouring the APO with the ctfA/B genes from C. acetobutylicum with the same strain harbouring APO with the ctfA/B genes from C. botulinum. Constructs were grown in TYG with 30 mM glucose with 1 ml samples being taken at 8, 24 and 48 hour timepoints. Gas chromotography was used to measure the acetone concentration in the supernatant.

Investigate the Obtainable Expression Range using an Inducible System

The PLAC system was used to determine the minimum and maximum BotR-dependent expression that could be achieved in our reporter strains.


Figure 24 shows a positive correlation between increasing concentrations of lactose, resulting in an increase in relative fluorescence of the PLAC strains harbouring the FAST reporter constructs under the control of the BotR dependent Pntnh promoter. There is a 98-fold increase in reporter expression from the uninduced state to the PLAC cultures induced with 10.0 mM of lactose. 


Figure 25 shows a similar positive correlation resulting in increasing GusA reporter activity as the concentrations of lactose increase. There is a 13-fold increase in reporter expression from the uninduced state to the PLAC cultures induced with 10.0 mM of lactose. This suggests there is less of a range in reporter expression with the gusA constructs than with the FAST constructs.


PLAC inducible expression of the APO was also assayed; however, there was a problem with the processing during the GC preparation step. Unfortunately, we were unable to analyse this data.


Based on this information, we made sure to test our electronic nose could detect a range of acetone concentrations with over a 98-fold difference between the highest and lowest concentrations.


Figure 24.

Expression range of C. sporogenes ΔpyrE::PLAC-botR harbouring the FAST reporter under the control of the BotR dependent Pntnh promoter. PLAC FAST constructs were induced with 0.1 mM, 1.0 mM and 10.0 mM of lactose at 8 hours.


Figure 25.

Expression range of C. sporogenes ΔpyrE::PLAC-botR harbouring the gusA reporter under the control of the BotR dependent Pntnh promoter. PLACgusA constructs were induced with 0.1 mM, 1.0 mM and 10.0 mM of lactose at 8 hours.

Switching On/Off Reporter Expression

We sought to further establish whether reporter expression in our engineered reporter strains could be influenced by the same factors that activate or repress toxin production in the native context of C. botulinum.


For instance, several publications reference the influence of arginine on repressing toxin synthesis in C. botulinum6,7. In this context, we replicated the protocol from Fredrick et al.[7], to investigate reporter expression in Toxin Production Medium (TPM) (2% w/v tryptone, 1% w/v yeast) supplemented with 0.5% w/v glucose (+ Glucose), 2% w/v arginine (+Arginine) or 0.5% w/v glucose and 2% w/v arginine (+ Glucose + Arginine). Since all three reporter constructs generated similar expression profiles, any of them could have been selected for this experiment; we chose to use the FAST reporter, as it offered the most reliable, time-saving and user-friendly way of measuring reporter expression.


Figures 26a-26c show the growth profiles of the engineered strains in the four different media, over the course of 48 hours. As expected, for all strains assayed, media containing higher nutritional content (i.e., supplemented with arginine, glucose or both) supported higher culture cell densities, compared to the basic TPM medium. It appears that the ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST strain attained slightly lower cell densities than the positive and negative control strains, in all media employed. It should be noted that the basal TPM medium is only half as rich in nutrient content as the TYG medium employed previously.


Figure 26a. Optical Density at 600 nm (OD600) of the engineered ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST strain, cultured in different media: toxin production medium (TPM); TPM + 0.5% w/v glucose (+ Glucose); TPM + 2% w/v arginine (+Arginine); TPM + 0.5% w/v glucose and 2% w/v arginine (+ Glucose + Arginine). OD600 measurements were taken at 0, 2, 4, 6, 8, 10, 24 and 48 hours.

Figure 26b. Optical Density at 600 nm (OD600) of the wildtype strain harboring the pMTL82151-Pfdx-FAST plasmid (+ve control), cultured in different media: toxin production medium (TPM); TPM + 0.5% w/v glucose (+ Glucose); TPM + 2% w/v arginine (+Arginine); TPM + 0.5% w/v glucose and 2% w/v arginine (+ Glucose + Arginine). OD600 measurements were taken at 0, 2, 4, 6, 8, 10, 24 and 48 hours.

Figure 26c. Optical Density at 600 nm (OD600) of the wildtype strain harboring the pMTL82151- FAST plasmid (-ve control), cultured in different media: toxin production medium (TPM); TPM + 0.5% w/v glucose (+ Glucose); TPM + 2% w/v arginine (+Arginine); TPM + 0.5% w/v glucose and 2% w/v arginine (+ Glucose + Arginine). OD600 measurements were taken at 0, 2, 4, 6, 8, 10, 24 and 48 hours.

Figure 27 shows a 2.2-fold decrease (p<0.0001) in FAST expression at 24 hours when the ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST strain is grown in medium containing arginine, compared to the control TPM medium, which is in favor of the role of arginine in repressing the toxin expression mechanism. Nevertheless, FAST expression of the pMTL82151-Pfdx-FAST construct was also lower when grown in arginine-containing medium (1.7-fold decrease; p=0.0051), indicating that arginine-induced repression of reporter expression may also be independent of botR. As expected, the negative control showed negligible levels of FAST expression, independently of the growth medium.


Figure 27. Effect of different growth media on FAST reporter expression. Expression of FAST in the indicated strains was assayed at 24 hours, after growth in different media: toxin production medium (TPM); TPM + 0.5% w/v glucose (+ Glucose); TPM + 2% w/v arginine (+Arginine); TPM + 0.5% w/v glucose and 2% w/v arginine (+ Glucose + Arginine). Relative fluorescence (RFU/OD) was obtained by dividing fluorescence by the respective OD600 values for each sample at the 24-hour timepoint. Data represent mean values of three of three biological replicates for all engineered strains ± SD. Statistical analysis was carried out using two-tailed unpaired t-tests; p-values are indicated as: 0.1234 (ns), 0.0332 (*), 0.0002 (***), <0.0001 (****).

Interestingly, addition of glucose also caused a significant decrease (5.7-fold; p<0.0001) in FAST expression in the ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST strain, but not in the strain harbouring the pMTL82151-Pfdx-FAST vector.

Quantitative Characterisation of FAST, Pfdx and other Clostridium promoters in E. coli and C. sporogenes

In order to share the quantitative characterisation of Bba_K2715011, BBa_J23106, and other promoters as useful to the iGEM community as possible, we first created the standard curves of the fluorescence signal measured by our plate reader relative to different standard fluorescein concentrations. This fluorescence per concentration of fluorescein was then converted to molecule-equivalent of fluorescein using the spreadsheet provided by iGEM measurement Hub (https://2019.igem.org/Measurement). A similar standard curve was calculated to correlate our absorbance measurements at 600nm to a number of particle (Cospheric Monodisperse Silica Microspheres 0.961 µm diameter).


The following standard curves were obtained (Figure 28a-28d):


Figure 28a.

Figure 28b.

Figure 28c.

Figure 28b.

Parameters were adjusted to increase the linear range of our standard curves. However, the particle standard curve was still only linear at higher absorbance values, so only the experimental data falling within this linear range were analysed.


Once the conversion factors from Arbitrary fluorescence units to MEFL and Abs600 to number of particles were determined, the quantitative assay of Pfdx, Bba_K2715011, BBa_J23106, and FAST could be achieved.


Figure 29. Relative fluorescence of E. coli FAST  constructs under the control of various promoters. The promoters: PbotR, Pntnh, Pthl, Pfdx, Bba_K2715011, BBa_J23106 and no promoter. Fluorescence expressed in Molecule equivalent of Fluorescein per Particle.

Figure 30. Relative fluorescence of C. sporogenes- FAST  constructs under the control of various promoters. The promoters: PbotR, Pntnh, Pthl, Pfdx, Pfdx Bba_K2715011BBa_J23106 and no promoter. Fluorescence expressed in Molecule equivalent of Fluorescein per Particle.

The first observation from the expression of the FAST protein using different Clostridium and E. coli promoters is that FAST is a suitable reporter gene, both in E. coli and in Clostridium sporogenes (Figures 29 and 30). Indeed, quantifiable levels of fluorescence were recorded in between 6.3*103 MEFL/particle and 1.1*106 MEFL/particle. However, it is worth noting, the assembly of the strong promoter BBa_J23119 with the FAST gene only produced colonies with heavily mutated BBa_J23119, indicating that FAST expression could be toxic under the control of such a strong promoter. It is however somewhat surprising that Pthl was not also mutated, as last year’s interlab study consistently found that Pthl was a stronger E. coli promoter than PJ23119.


Pfdx and Bba_K2715011 have equivalent expression levels (5.5*105 +- 0.9*105 MEFL/particle and 6*105 +-1*105 MEFL/particle respectively in C. sporogenes; 9*105 +- 1*105 105 MEFL/particle and 1.007*106 +-0.009*106 105 MEFL/particle respectively in E. coli). As such, Pfdx and Bba_K2715011 are interchangeable. However, since our part “Pfdx” is the original, unmutated version of the ferredoxin promoter from C. sporogenes, the sequence of Bba_K2715011 documented in the registry of parts should be curated to match the native sequence of Pfdx.


Additionally, BBa_J23106 is being expressed at significant levels (p<0.05) in C. sporogenes, with a normalised fluorescence of 6.3*104 +-1.3*104 MEFL/particle. Consequentially, this promoter could be used in the future for genes that require very low expression in C. sporogenes. Similarly, Pntnh and PbotR exhibit low but significant expression in C. sporogenes, proving that they can be used in the assembly of our reporter constructs.


Interestingly, all promoters seem to be much more strongly expressed in E. coli than in C. sporogenes.

References

1. Koransky, Jack R., Stephen D. Allen, and V. R. Dowell Jr. 1978. "Use Of Ethanol For Selective Isolation Of Sporeforming Microorganisms". American Society For Microbiology 35 (4): 762 - 765.
2. Millat, Thomas, Holger Janssen, Hubert Bahl, Ralf-Jörg Fischer, and Olaf Wolkenhauer. 2013. "Integrative Modelling Of Ph-Dependent Enzyme Activity And Transcriptomic Regulation Of The Acetone-Butanol-Ethanol Fermentation Ofclostridium Acetobutylicumin Continuous Culture". Microbial Biotechnology 6 (5): 526-539. doi:10.1111/1751-7915.12033.
3. Bahl, Hubert, Wolfram Andersch, and Gerhard Gottschalk. 1982. "Continuous Production Of Acetone And Butanol By Clostridium Acetobutylicum In A Two-Stage Phosphate Limited Chemostat". European Journal Of Applied Microbiology And Biotechnology 15 (4): 201-205. doi:10.1007/bf00499955. https://link.springer.com/article/10.1007/BF00499955
4. “Botulism Associated With Commercial Carrot Juice --- Georgia And Florida, September 2006". 2019. Gov. https://www.cdc.gov/mmwr/preview/mmwrhtml/mm55d106a1.htm
5. Inés C. Cañadas, Daphne Groothuis, Maria Zygouropoulou, Raquel Rodrigues, and Nigel P. Minton 2019. RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium ACS Synthetic Biology2019 8 (6), 1379-1390 DOI: 10.1021/acssynbio.9b00075
6. Clostridium botulinum Okra B and Hall A by arginine. Appl Environ Microbiol, 1989. 55(6): p. 1544.
7. Fredrick, C.M., Lin, G., and Johnson, E.A., Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502. Appl Environ Microbiol, 2017. 83(13).
8. Johnson, E.A. and Bradshaw, M., Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective. Toxicon, 2001. 39(11): p. 1703-22.