Construct part extraction from E. coli cultures

Growing E. coli cultures for extracting construct parts

Cultures for all gene fragments from the library were made: P0 backbone, P1 promoter, P2 RBS S3, P2 RBS S4, P3 sYFP2, P4 RBS S3, P4 RBS S4, P4 RBS S5, P5 EFE and P6 terminator. Antibiotic selection was conducted with spectinomycin and ampicillin.

Preparing glycerol samples of construct part E. coli strains

Glycerol preps were made from the E. coli cultures. The samples were placed in the freezer (-80°C) for later use.

Making plasmid preparations of E. coli cultures

Plasmid preparations were made from E. coli cultures using either the Monarch Plasmid Miniprep Kit (NEB) or Qiagen Midiprep kit (pDF and promoter) following the protocols provided by the manufacturers.

Preparing the construct assembly

Preparing competent E. coli cells for transformation

Competent cells were prepared for transformation by making a culture with inoculated DH5α E. coli cells. The culture was centrifuged, the pellet resuspended with CaCl₂ and stored in the freezer (-80°C) for later use.

Preparing commercial genes

We ordered the merA (from Synechocystis and Pseudomonas aeruginosa) and merB genes (E. coli and Pseudomonas aeruginosa) from IDT and Twist. The genes were prepared following the instructions provided by the manufacturers.

Construct assemblies and screening

Modified Golden Gate assembly

In our experiments we used a modified Golden Gate assembly to create plasmid constructs with different combinations of our merA and merB genes and different ribosome binding sites (RBS). We made several assemblies with this one-pot Golden Gate reaction with all the fragments in a single tube in order to construct several complete plasmids simultaneously. The general structure of our plasmids was as follows: starting with a plasmid backbone, then a promoter at position 1, RBS at position 2, one of our merA homologs at position 3, another RBS at position 4, one of our merB homologs at position 5 and a transcription terminator at position 6.

For each assembly, we also prepared positive controls by replacing our genes of interest with sYFP2 and EFE. Negative controls were also prepared by using only the plasmid backbone. Later we also tried creating assemblies with only one gene of interest, while the other was replaced with either sYFP2 or EFE depending on the position of the replaced gene.

For the assembly reaction, two mastermixes were prepared: a reagent mastermix with all the necessary PCR reagents and a fragment mastermix. The fragment mastermix was prepared with our genes of interest, RBSs, promoter, transcription terminator and plasmid backbone. The genes and ribosome binding sites were different in each assembly reaction and the amount of each varied between assemblies due to different concentrations. A digestion ligation cycle (RecBCD) was run on the samples.

Heat-shock transformation (E. coli )

PCR samples were transformed into the competent DH5α E. coli cells using heat-shock. DNA-cell samples are briefly exposed to a high-temperature and then immediately placed back on ice. This method is based on the principle of thermal stress which makes the bacteria more likely to accept foreign DNA. The control included a plate with only the backbone as well as a plate with everything else except backbone verifying the success of transformation.

Colony screening by PCR (E. coli )

After a successful transformation the possibility of positive results were high. To verify that our assembly had worked and that we had successfully constructed modified plasmids, we did colony screening by PCR. The forward primer in the PCR binds towards the end of the promoter and the reverse primer to the other end of the pDF vector. This means the PCR covers the entire length of all inserts as well as a small portion of the promoter and pDF backbone. E. coli samples were randomly selected colonies from our LBA plates. The amount of samples varied between transformations, but the PCR master mix was always prepared 15% more than our sample size.

Agarose gel electrophoresis (E. coli )

The PCR results were studied with 1% agarose gel electrophoresis with positive and negative controls.

Growing Synechocystis

Making a liquid culture of cyanobacteria

Liquid culture was made from Synechocystis sp. PCC 6803. The cyanobacteria were flushed from a growth plate with BG-11 medium. The culture was put in a growth chamber where the main liquid cultures were made from the previous one with a starting OD750 of 0,1A

Making Synechocystis sp. PCC803 storage cells

Once the absorbance of the liquid culture had reached 0,597 A at 750 nm. Glycerol preps were made from the Synechocystis culture. The samples were frozen with liquid nitrogen and put in the freezer at -70 °C for later use.

Making Synechocystis sp. PCC6803 BG-11 growth plates without antibiotics

BG-11 growth plates were prepared with bacto-agar. Samples were then plated with no antibiotics and put to grow in a growth chamber.

Synechocystis transformation and screening (future experiments)

Synechocystis sp. PCC6803 transformation

We have a modified transformation protocol based on the findings of Zang et al., and Lauri Kakko’s, a doctoral researcher at the Molecular Plant Biology unit of University of Turku, modifications. In this protocol EDTA was added to the liquid main Synechocystis sp. PCC6803 before the culture reached a mid logarithmic growth phase. The cells were plated on BG-11 plates first with no antibiotics. After a green film forms the antibiotics are added underneath the agar. Then the plates are covered with a paper sheet to reduce light intensity. The goal of all of these modifications are to add efficiency and reduce the growth and selection period of Synechocystis.

Colony screening by PCR and gel electrophoresis (Synechocystis)

The colony screening with Synechocystis includes freeze-thaw cycles.

The protocol for gel electrophoresis is the same as above with E. coli DNA samples.

Mercury experiments (future experiments)

To test the function of the Synechocystis sp. PCC 6803 cell strains that have been transformed with merA and merB genes, our idea was to perform mercuric resistance experiments on the modified cyanobacteria. The ability of the modified cells to grow in the presence of mercury compounds can be used as an indicator of the function of the merA and merB genes. Our team did not have time to perform these experiments with offers a unique opportunity for further research. The protocols for mercury resistance experiments would have been based on the protocols by iGEM team Minnesota 2014 (Mercury(II) Chloride Plate test protocol - parts.igem.org, n.d.). The protocols would have been slightly modified and adapted to our cyanobacteria-based system.

Our plan was to make two different assays to test the function of merA and merB genes separately. In the experiments, we would have prepared agar plates with a filter disk spotted with methylmercury or mercury(II) chloride in the middle of the plates. In one assay, we would make agar plates with 10µL of 0.1M methylmercury on a filter disk to screen the function of the merB gene. The diameter of the area where the cells can not grow would have been measured and compared between different cell strains. The plates would have contained cells with constructs that either include the merB gene alone or both merB and overexpressed merA. The mercury resistance of modified cyanobacteria would have been compared to unmodified cyanobacteria. Because the assembly was performed in E. coli , we would also have tested and compared the function of the constructs in E. coli versus Synechocystis sp. PCC 6803.

In the other assay, we would have made agar plates containing 10µL of 0.1M mercury(II) chloride on a filter disk to test the function of the merA gene. The mercury(II) chloride resistance of Synechocystis with overexpressed merA would have been compared to unmodified Synechocystis that has a natural merA gene. The results would also have been compared with E. coli cells with the merA containing plasmid construct.

Later, had the constructs been qualitatively shown to function in the resistance assays, we would also have wanted to test the constructs quantitatively using ICP-MS to measure the changes in methylmercury and mercury ion concentrations. This would have required preprocessing of the samples since ICP-MS only can detect the total concentration of mercury.