After the search for genes came the part of designing the genetic circuit. Having several teams already used the J23118 promoter for their experiments we chose to use this medium strength promoter along with the strong RBS (B0034) and call it the medium strength expression system. This system was designed to be used along with all the genes in the system except DNaseI which was designed downstream to the araBAD promoter , which is an arabinose inducible promoter. All these systems were mathematically modelled and simulated for their theoretical expression as we predicted their behaviour.
Check out our Project Design and Model page to know more!
Design of Future Experiments
Quantification of sulfonamides in animal excreta
Estimating the concentration of sulfonamides is crucial for determining the amount of enzymes required and the degradation time. This can be obtained by running the samples through the HPLC column and then comparing it with standard plots for sulfa drugs.
The collected excreta samples will be oven-dried at 70℃ and then powdered. A set of samples will be prepared by dissolving known concentrations of the powdered excreta in the solvent (methanol). This gives a range of samples varying from extremely diluted to concentrated. These excreta samples will then be filtered using a PVDF filter and run through the HPLC column following the standardized method (Check out the Protocol page). The output data for different samples will be plotted as graphs and will be compared with the standard plot to get the concentration. The formula for calculating the concentration from peaks heights (PH) is as follows:Cstandard /PHstandard = Cunknown/PHunknown
= concentration of standard sulfa drug dissolved in methanol
= concentration of excreta sample dissolved in methanol
= Peak height of standard sulfa drug dissolved in methanol
= Peak height of the excreta sample dissolved in methanol
Once the concentration for different samples is calculated, the error bar will be determined to get the range of the sulfonamides. This will be further used for sulfonamide degrading enzyme kinetic calculations.
The genes sulX and sulR will be cloned into the vectors pSB1C3 and pSB3K3 respectively and will be transformed into calcium chloride competent DH5α cells individually in the presence of respective antibiotics. The colonies will be screened for positive clones, thereafter the colonies will be cultured followed by plasmid isolation. The recombinant plasmids will then be co-transformed into competent BL21 cells. Similar cloning strategy will be followed to clone traS, traT and DNaseI into the same bacterium.
Action of sulfa drugs on the engineered bacteria
The effect of different sulfa drugs on the clone and non-resistant control bacteria will be measured by broth assays. Both bacteria will be cultured in media containing different sulfonamides and the survival advantage of the clone will be measured over the control bacteria. Following this, it will be measured if sulX or sulR when present alone in the bacteria provide survival advantage and this will be quantified. To experimentally show this, individual clones expressing SulX or SulR will also be generated. Broth assays and disk-diffusion assays with different concentrations of sulfonamides will be conducted and quantified wherever possible. The experiments will be repeated at least three times and the required statistical tests would be carried out. We expect to see a significant difference between the survival of clones with both genes (containing both sulX and sulR) and clones containing either sulX or sulR.
Quantification of the rate of sulfonamide degradation
The clone will be cultured in different concentrations of sulfonamides at different environmental conditions in minimal media. This media will be collected at regular intervals and analysed by the HPLC method developed to quantify sulfonamide degradation by the clone. The methods for detecting sulfonamides have already been established as a part of preliminary feasibility study (Check out our Protocol page).
Determining the percentage reduction in conjugal frequency
The conjugal donor strain MFDpir containing the part K873003 will be mated along with our bacteria (expressing TraS and TraT, containing two fluorescence markers on either of the plasmids) in appropriate antibiotic containing media and the transconjugants will be selected based on fluorescence and donor’s antibiotic resistance marker. The Mating will be done separately for engineered bacteria and control strain. The effective colonies would be positive for both the antibiotic markers and should express both GFP and RFP simultaneously.The final reduction in conjugal transfer will be calculated using the formula below-
Reduction in conjugal transfer =
(control recipients - engineered recipients)*100/control recipients
Inducible expression of bpDNaseI and its purification
DNaseI is used here as a death-inducing gene that cleaves DNA into smaller fragments thus inducing cellular death. This gene will be cloned downstream to the tightly regulated araBAD promoter into both the vectors. This will be transformed into BL21 cells. These cells will be stimulated with the appropriate arabinose concentration and cell death will be measured by monitoring the OD of the culture. The culture will be plated at regular intervals to determine the CFU. Also, DNA from such cultures will be isolated and run on an agarose gel to check for any intact DNA prevailing. The bpDNaseI will also be overexpressed and will be purified for in-vitro characterization to determine its DNA degrading efficiency.