Team:Nottingham/Demonstrate


Project

Demo



After months of troubleshooting and hard work, our mutants and the electronic nose have finally come to life!

Project
Demo

Accurate Simulation of Botulinum Toxin Expression using Reporter Constructs in C. sporogenes

By placing plasmid-borne reporter expression under the control of a BotR cognate promoter (Pntnh), we were able to show that functional reporter expression can be obtained in C. sporogenes, only in the presence of BotR. This mimics the specific way in which botulinum toxin is elaborated in the wildtype C. botulinum strain. In fact, BotR-mediated activation of Pntnh was highly specific for all three reporters considered, as evidenced by the lack of reporter expression from plasmids bearing the Pntnh-reporter constructs, when conjugated in the wildtype strain (lacking BotR). This is shown in the FAST, GusA and acetone expression graphs on the Results page.


Importantly, in our reporter strains (ΔpyrE::PbotR-botR + pMTL82151/pMTL82121-Pntnh-reporter), an increase in reporter expression was observed during the late exponential/death phase, paralleling the profile of toxin production in C. botulinum, as shown in Figures 2-4. This was true for all reporters studied and provides further evidence that BotR-dependent transcription of reporter genes in C. sporogenes occurs in a similar way to the transcription of the toxin genes in C. botulinum.


Figure 1. BoNT-A toxin concentration in culture supernatants of the C. botulinum ATCC 3502 wildtype strain, over time, alongside the corresponding OD600 of the cultures.[1]
Figure 2. FAST expression (left y-axis) in the ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST strain, over time, alongside the corresponding OD600 of the cultures (right y-axis). Three biological replicates of the strain were grown in TYG with 30 mM glucose and 1 ml samples were taken at 8, 24 and 48 h timepoints. Relative fluorescence (RFU/OD) was obtained by dividing fluorescence by the respective OD600 values at each timepoint.

Figure 3. Acetone concentration (left y-axis) in culture supernatants of the ΔpyrE::PbotR-botR + pMTL82151-Pntnh-APO strain, over time, alongside the corresponding OD600 of the cultures (right y-axis). Three biological replicates of the strain were grown in TYG with 30 mM glucose and 1 ml samples were taken at 8, 24 and 48 h timepoints. Acetone was determined in culture supernatants using GC.

Figure 4. Normalised FAST reporter activity of ΔpyrE:PbotR + pMTL8215x-gusA. Constructs grown in TYG with 30 mM glucose with 1ml samples being taken at 0,4,8,24 and 48 hour intervals. Samples were assayed over 30 minutes to measure gusA reaction rate. Data was normalised by dividing fluorescence by OD600.

We sought to further establish whether reporter expression in our engineered reporter strain could be influenced by arginine, a well-established repressor of toxin production in C. botulinum.[2][3] Indeed, we found that for our ΔpyrE::PbotR-botR + pMTL82151-Pntnh-FAST reporter strain, addition of arginine in media yields a 2.2-fold decrease (p<0.0001) in FAST expression at 24 hours. Nevertheless, a smaller but also statistically significant decrease in FAST expression also occurred for the pMTL82151-Pfdx-FAST construct (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 5. 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. This is contrary to previously published data suggesting that addition of glucose in media has a stimulatory effect on toxin production, as observed in the C. botulinum ATCC 3502 strain.[3] Despite employing the botR and Pntnh sequences from this exact strain, this contrasting observation may be due to differences arising in the genetic context of C. sporogenes vs that of C. botulinum, which may be influencing reporter expression in unknown ways. In fact, toxinogenesis patterns in response to the same metabolic stimuli may have marked differences even between closely related C. botulinum strains.[4] As a result, further investigation is warranted to obtain a deeper understanding of toxin regulation in our reporter strain. Ultimately, such differences may not be relevant, given that the final Notox strain is envisaged as a safe strain of C. botulinum. 

Acetone Detection by our Self-Designed Electronic Nose

We prepared acetone standards covering a 100-fold concentration range, using the information we obtained from our reporter strains with inducible expression of botR (ΔpyrE::PLAC-botR + pMTL82151-Pntnh-reporter).


Known acetone concentration (mM)

0

0.5

1

2

5

10

20

50

100

Average of 3 readings (mV)

110

195

135

127

427

646

664

805

896

Average of 3 readings (mM)

0.5

2.20

1.01

0.85

7.89

18.8

20.4

43.2

109.5

Figure 6. Comparison of known acetone concentration with the concentration detected by the electronic nose’s sensor.

According to the manufacturer, the sensor detects acetone between 0.86-86 mM. We chose a range of acetone concentrations, some of which were deliberately outside of the sensor’s range. The results show the sensor is able to detect accurately concentrations of acetone between 1-50 mM. The 2mM acetone concentration was not accurately detected, possibly due to a pipetting error. Small deviations observed between the actual and nose-recorded concentrations may be due to humidity and temperature fluctuations. Implementing an enclosed sensor design may help overcome this shortcoming.

References

1. Blount, B., Analysis of the roles and regulation of flagella in Clostridium botulinum. 2011, University of Nottingham.
2. Patterson-Curtis, S.I. and Johnson, E.A., Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine. Appl Environ Microbiol, 1989. 55(6): p. 1544.
3. 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).
4. Johnson, E.A. and Bradshaw, M., Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective. Toxicon, 2001. 39(11): p. 1703-22.