After all genes were successfully able to be amplified with the specific homology regions (either IVA or SLIC), chunks of genes were assembled. Since nine genes make up the total operon, we attempted to split the genes into triples. Thus, nifB-nifH-nifD were assembled as one cluster, nifK-nifE-nifN as another cluster, and nifX-hesA-nifV as a final cluster. Both IVA and SLIC methods were used to approach these intermediate assemblies. Protocols directly from the original IVA or SLIC papers were closely followed. Any deviations from the protocol are detailed under the protocols page.

After the three intermediate segments were assembled, the complete assembly of the operon was attempted. Primers to attach homology regions to the segments and plasmid 2Bc-T were used to amplify the segments. Because more success was achieved with the in vivo assembly method, this method was pursued primarily. All cloning results were screened initially with colony PCR or single restriction enzyme digests. Final screening was verified through whole plasmid sequencing through a third-party sequencing service, Plasmidsaurus. See our results page for our sequencing results and analysis!

Concurrently to the assembly of the full nitrogenase operon, a plan to integrate the full operon in place of the E. coli gene araC was developed. Firstly, we chose this target region because we wanted to accomplish the following: (a) to knock out araC, which would deter predictability of the oscillation cycles according to our dry lab by expressing in baseline levels and (b) to knock-in the operon in a well-characterized region of the genome with complete control over copy number. Based on papers we found on multigene-editing through lambda-red/CRISPR coupled technology we designed a plan (see below figure) to integrate our assembled system into the E. coli genome [3,4].

Due to limited time, a coomassie stained gel was initially used to determine protein expression within the nitrogenase operon. (See figure on how coomassie works). This qualitative method involved over-expressing our nitrogenase genes through addition of IPTG, inducing for a range of times, and running the protein lysates through a SDS-PAGE. Then, a dye known as coomassie blue was added to the gel for subsequent staining. The stain, which selectively binds to certain amino acids, making it useful for estimation of protein concentration in a sample. See the below figure for an overview on how coomassie staining works or check out our protocols page for further information!

A more phenotypically approach was also pursued. Based on past iGEM team’s research, several nitrogen-deficient medias were evaluated. Our rationale for this assay was the following: if our engineered E. coli were able to survive in nitrogen-deficient media, then they must be expressing the enzyme, and the environment must be selecting for these phenotypes.

First, we wanted to confirm our models matched up with the Stricker et. al paper by expressing the dual-plasmid system (activator and repressor plasmids) under a confocal microscope. We wanted to show, as a proof of concept, that there was oscillation observed under our laboratory conditions.

Second, plans to clone the hybrid promoter as developed by Stricker et. al into the assembled cluster was established. The plan involved replacing the T7 promoter found at the beginning of 2Bc-T with the hybrid promoter using in-vivo assembly methods. This cluster would also be attempted to integrate into the genome, as we believed that would provide the most flexibility for controllable expression while also protecting against selective pressures that would cause species with the nitrogenase enzyme system to lose its parts in the wild.