Experimentation

Bacterial transformation

These are the experiments carried out from the instance we received the synthesized operons to when we transformed our bacterium.

I. Stock preparation

We received the genes in custom clonal vectors pMPV-alpha (ampicillin-resistant cloning and shuttle vector with pUC origin of replication for E. coli) from TWIST Bioscience. We first plated them in ampicillin-infused agar plates, where they responded favorably and grew quite well.

Then, we prepared primary and secondary glycerol stocks of all the plasmids and bacteria for future use.

II. Plasmid isolation

We used the QIAGEN Spin Miniprep kit to perform isolation of the pMPV-alpha-nirBD and pMPV-alpha-nrfHA, pMPV-alpha-nrfABCD, and pMPV-alpha-nrfEFG plasmids.

III. Restriction enzyme digestion

We designed the operons and chose plasmids such that separate DNA fragments would recombine after ligation to form the functioning operons once again.

Image source: Biorender (Created in BioRender.com)

Our restriction enzymes of choice are EcorI, BamHI, and HindIII.

For nirBD, the self-containing operon, the process would be simpler. Here’s a schematic of the plasmid creation process using pSEVA 648.

Image source: Biorender (Created in BioRender.com)

From left to right: 1-10kb standard DNA ladder, undigested plasmid, digested plasmid. No clear bands were obtained after digestion.

From left to right—Ladder, Undigested pSEVA648, pSEVA648+EcoRI, pSEVA648 + BamHI, pSEVA648+ HindIII. Only the HindIII digestion seemed to have worked, which prompted us to replace the EcorI and BamHI enzymes we were using.

Having replaced our stock with fresh enzymes, we performed digestion for all of our samples.

IV. Elution

We eluted the bands from the gel using a gel elution kit, which yielded less than satisfactory DNA concentrations yet again.

Since we had 50μL of the eluted sample, we used a vacuum heater to evaporate the excess supernatant and proceeded with ligation.

V. Ligation and Transformation

Our next step was to ligate the different fragments together. We picked nrfABCD and pSEVA648 for the first run due to the promising DNA concentrations obtained after drying the elution samples.

After setting up ligation and incubating, we transformed E. coli DH5-alpha using heat shock. Unfortunately, we did not observe growth on the Kanamycin agar plate, indicating that something failed either during ligation or transformation. We are currently troubleshooting the problem.

Identifying Optimal Growth Media

We prepared rich growth media and minimal growth media having varying compositions based on the questions brainstormed during the design phase of this cycle. The idea was to confirm which culture media P. putida KT2440 responded most favorably to, and then run further tests on nitrate and nitrite metabolism in the short-listed media for convenience. We also cultured E.coli DH5-alpha and P.putida EM42 to act as negative controls for the subsequent experiment.

We first took inventory of the material we had in our lab to prepare the media.

Media & Components

Compositions Table

Contains values for rich media

Minimal Media Table

Contains values for minimal media

Results

Triplicates of 200μl bacterial cultures were taken in 96-well microtiter plates for ease of processing. Their optical density (OD) values were subsequently measured at 650 nm using a Tecan Infinite® 200 PRO microplate reader.

Growth of P. putida KT2440 in various growth media

For P. putida KT2440, OD values were measured after 12, 24, and 48 hours of inoculation. The resulting data has been represented in the graph above. Samples 1, 5, and 6 showed the highest readings. Initially, sample 5 had the highest reading; however, it decreased at 24 hours and then slowly started increasing again. Sample 6 readings skyrocketed after 24 hours of incubation, whereas those of sample 1 increased steadily throughout. We can conclude that sample 5 is optimal for sub-24 hours of incubation, and sample 6 can be opted for longer incubation periods.

Both P. putida EM42 and E. coli DH5-alpha were incubated for 12 hours each. Their OD readings are colour-coded from highest to lowest.

Once again, sample 6 shows the highest reading, followed by samples 1 and 5.

E.coli DH5-alpha

Sample 5 shows the highest reading, with sample 6 close behind.

For all three bacterial species, samples 3, 7, and 8 showed very poor growth (often negative readings). The bacteria were cultured in samples 1, 5, and 6 for further testing.

Nitrite Metabolism in P. putida

To confirm the success of our engineered plasmid, we need to ensure that nitrite uptake and extracellular ammonium production of the transformed bacterium are higher than the basal levels already present in our chassis. The first step was thus to calculate the basal level of nitrite reduction already carried out by the bacteria.

We used samples #1, #5, and #6 to culture P. putida KT2440 and measured nitrite values at 12 hours, 24 hours, and 48 hours. We also cultured our negative controls to measure against a standard.

Growth of P. putida in various growth media

We used the Merck Nitrite Test, colorimetric, 0.005-0.10 mg/L (NO₂^-), 0.0015-0.030 mg/L (NO₂-N) assay kit to quantify nitrite concentrations. The recorded concentrations have been converted to g/mL for consistency with other related data.

The results indicate that the bacterium took up a significant amount of nitrate in just 12 hours. The nitrate levels in the supernatant fluctuated minorly for the 12, 24, and 48 hour readings. These trends are observed in all three cases, sample 1, sample 5 and sample 6.

The uptake of nitrate clearly indicates that DNRA/ANRA may be occurring. This matches our expectations since P. putida KT2440 has nitrite reductase operon nirBD even natively. We also conducted ammonium quantification assays to verify the reduction to ammonium, and whether it is DNRA or ANRA.