For future iGEM teams who wish to work with the oscillator, we wish to share the following results that were established through our experimentation with the plasmids that may inform or enhance their experimentation:

Low Copy Numbers

Both origins of replication for pJS167 and pJS169 have low copy numbers. pJS167 is a pACYC-derived plasmid with an origin of replication p15A. pJS169 has a ColE1 ori. Both origins have a copy number of around 10-15, depending on various factors such as growth media used, properties of insert in the plasmids, or incubation temperature [1]. We found that following several recommended guidelines, as suggested by commercially available kits helped increase the yield of plasmids significantly, and we have summarized and detailed those guidelines for future iGEM teams. See our experiments page to see a specific protocol tailored for low copy plasmid minipreps.

Design Limitations

pJS167 is an interestingly designed plasmid in that there are multiple regions before and after both the GFP gene and the araC gene that are identical in sequence. We considered the possibility that future iGEM teams may want to alter pJS167 to be tailored to their own purposes. While restriction enzyme-free cloning would be an attractive approach to doing so, since assembly approaches such as Gibson Assembly or HiFi Assembly have been very popular amongst competitors, the nature of the plasmid makes it very difficult to design primers needed for these methods. Our original plan was to cut out GFP to place the nitrogenase cluster in place of it but found that primers designed to anneal near the GFP coding region were unspecific, and no unique restriction enzyme sites near the sites. We recommend that designing around pJS167—by one of three ways: (1) editing pJS169 (2) assembling a separate plasmid with araC, GFP, and the gene/modification of interest or (3) inserting the hybrid promoter in front of the target modification and inserting the whole system into a separate vector or the genome.

Designs Using Nitrogenase

While we ultimately decided that integration into the genome would be the most stable way to express our system, we considered incorporating nitrogenase into one of the dual plasmids themselves. Due to the design limitations documented in pJS167, pJS169 was considered as a potential plasmid to insert our nitrogenase cluster in. Although this plan was never officially pursued, primers to insert the cluster with SLIC methods were designed. Our work will help guide future teams who wish to insert a gene under the control of the oscillator, and are considering editing the repressor plasmid to do so.

Nitrogenase Cluster

Segments of nif genes with IVA

As mentioned in our experiments page, we assembled our nitrogenase operon dividing the nine individual genes into three assembled segments first. While both SLIC and IVA methods were attempted, we ultimately decided to use IVA to assemble the three segments — nifB-nifH-nifD, nifK-nifE-nifN, and nifX-hesA-nifV.

We believe that successfully assembling these three segments will make assembly of the full operon much easier for future teams. Nitrogenase is a popular enzyme amongst iGEM teams, but there is no standard part in the iGEM registry that can be distributed to future teams. Our assembly of the three parts, if requested altogether, can immediately be used for IVA in an afternoon to amplify the segments using PCR, followed by a DpnI digest, and finally transforming into most laboratory strains of E. coli cells to observe and screen for positive colonies in the morning.

Our sequence parts are now registered in the iGEM registry with the part names: BBa_K4647029, BBa_K4647030, and BBa_K4647031. When synthesizing our parts through TWIST and/or IDT, we performed silent mutations of the four illegal and incompatible restriction enzymes for BioBrick assembly with SnapGene’s silent mutation software. We found an average of 1-2 of illegal restriction enzyme sites per gene in the cluster characterized by the 2018 Nanjing China Team (BBa_K2740011). Our clusters will be biobrick compatible for future teams who wish to use a different method of assembly than we have.

We would like to thank the 2018 Nanjing China Team for providing us with the minimal nif cluster sequence from Polymyxa CR1. Their work on characterizing the nif cluster served as the foundation of our project. We hope our work will allow future teams to be able to work easier with nitrogenase.

In Vivo Assembly

As far as we know, there has been no iGEM team yet that detailed using IVA as an assembly method for an insertion of our size. We chose this method for a few reasons: ease of access, less hands-on work time, and low cost.

In vivo assembly is a cloning technique that is both scarless and homology-directed repair based. The methodology was developed by García-Nafría and Watson et. al in 2016 [2]. The method takes advantage of the fact that there is a recA-independent pathway capable of joining homologous regions and repairing them within E. coli itself— thus eliminating the step to transform assembled parts into E.coli (or desired host).. Although the mechanism is not yet elucidated completely, the paper demonstrated high experimental success with the cloning technique. They show the technique is very flexible as well, capable of performing insertion, deletions, subcloning, and multi-site edirected mutagenesis depending on how the primers used are designed.

While there are extensive advantages of using IVA, there are some drawbacks to the technique that we uncovered through experimentation. For further elaboration on these points, please refer to our experimentation and results pages!

Sequence and Ligation Independent Cloning

Sequence and Ligation Independent Cloning, or SLIC is another technique that can be used in place of other popular methods of cloning techniques. The biggest difference between SLIC and IVA is that SLIC is an in-vitro technique, while IVA is in-vivo. We were unsure of the efficiency that IVA would be able to stitch together genes, so SLIC and IVA were pursued concurrently.

SLIC was first detailed by Li and Elledge et. al in 2012 [3]. The method, very similar to ligation independent cloning [4], involves attaching homology regions of desired length to individual DNA segments. The technique then takes advantage of T4 exonuclease activity and allows the enzyme to “chew back” the sequence, creating sticky ends. A ligation step follows where homologous regions are joined together by their sticky ends. The assembled plasmid is then transformed into a host.

We found that SLIC had potential advantages over IVA, but overall, IVA was a more direct approach for our cloning needs. However, both protocols are documented in great detail so future iGEM teams could evaluate which technique better suits their needs.

Please refer to our experimentation page, protocols, and primer documentation page for more details on these above points!

Leica Confocal Imaging for the Genetic Oscillator

We worked on replicating the 2008 Stricker oscillator, by co-transfecting the dual-plasmids into the expression strain JM109 DE3 and observing the resulting GFP signals on a confocal microscope. We were given access to a Leica SP5 model microscope generously by Dr. Peter Gergen’s laboratory staff, Yasuno. She helped us by training our team leaders in working with the confocal, troubleshooting our results, and analyzing some results by suggesting possible interpretations.

The specifications were E. coli were detailed in our protocols, in the experimentation section. Check our results page as well for the changes in GFP signal we saw, protocols, and possible troubleshooting and analysis we have to perform. We hope that our work would help inform other teams who want to image E. coli with a confocal microscope.

References

1. Stricker, J., Cookson, S., Bennett, M. R., Mather, W. H., Tsimring, L. S., & Hasty, J. (2008). A fast, robust and tunable synthetic gene oscillator. Nature, 456(7221), 516–519. https://doi.org/10.1038/nature07389


2. García-Nafría, J., Watson, J. F., & Greger, I. H. (2016). IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly. Scientific Reports, 6(1). https://doi.org/10.1038/srep27459


3. Li, M. Z., & Elledge, S. J. (2012). SLIC: A method for sequence- and ligation-independent cloning. Methods in Molecular Biology (Clifton, N.J.), 852, 51–59. https://doi.org/10.1007/978-1-61779-564-0_5


4. Aslanidis, C., & de Jong, P. J. (1990). Ligation-independent cloning of PCR products (LIC-PCR). Nucleic acids research, 18(20), 6069–6074. https://doi.org/10.1093/nar/18.20.6069