Engineering Success With Reporter Genes

A reporter gene is a coding sequence that makes an easily identifiable protein. The wide variety of fluorescent proteins are all great examples of reporter genes. Throughout our iGEM project, we have been building plasmids with three different reporter genes.

GUS, originally designed by a PhD student at our university, is a popular reporter gene to use for transgenic organisms. A gus reporter gene codes for a β-glucuronidase protein that can hydrolyze a clear substrate into a visible blue one. This gene works in a similar way to the β-galactosidase enzyme that cleaves X-Gal to produce blue colonies in E. coli. Early in the project, we began building a plasmid to add a promoter and a terminator to the GUS coding sequence. However, we had problems getting our E. coli cells to grow on plates after electroporation. It was not until we went back and adjusted our electroporation protocol to include tapping out air bubbles in the electroporation cuvette that we were able to successfully transfect our E. coli cells. While we worked on these plasmids, there was still a background issue; a problem with GUS as a reporter gene is that staining to see where GUS is present and expressing requires sacrificing our plant cultures.

Eventually, our lab turned to fluorescent proteins as a means of identifying transformation efficiency. While there are many popular fluorescent proteins, our lab chose to use a superfolder green fluorescent protein based off of previous work on soybeans including work by Yang et al. 2019¹. We constructed several plasmids that included this GFP reporter genes, including a Gmubi*CrtE-cytoTDS-MBP*AtHSP*Gmubi*GFP*AtHSP. When tested, however, we learned that our means of identifying GFP allowed for a lot of background signaling to be present due to UV excitement and autofluorescence of native soybean molecules. This led to an overestimation of correctly transformed cells and made it harder to decide what parts of the plant should be analyzed for expression.

Unfortunately we did not have the means to improve our system for viewing GFP, so we had to try another design to make our reporting system work. This led us to RUBY². RUBY converts the amino acid tyrosine into betalain, the pigment that makes beets and swiss chard appear red. After adding a promoter and terminator, RUBY is translated and transcripted before undergoing two self mediated cleavages into three different proteins.

In-lab experimentation showed promising results for RUBYs use as a reporter gene in soybean. To the naked eye, it is possible to see clumps of RUBY expression in soybeans that have undergone agrobacterium mediated transformation.

One of our final plasmids, “CrTE-cytoTDS-MBP_RUBY (BBa_K4201016), where both genes were promoted with Gmubi and terminated with AtHSP, was ready in time to test for the iGEM competition. After sequencing confirmed our plasmid had been correctly assembled, it was moved into agrobacterium and replicated following the protocol for agrobacterium transformation. Next, it was introduced into plants following the Plant Transformation Protocol including the optional additions of DTT and cysteine to the infection and co-cultivation medium. The addition of these strong reducing agents to the infection and co-cultivation medium help to suppress the soybean's immune response to agrobacterium and may increase transformation efficiency. On day seven (October 7th, 2022), prior to movement from the plate to the culture vessel, expression of RUBY was observed. Prior in-lab tests of RUBY without the addition of reducing agents first saw visible signs of RUBY on approximately day 14. As the tissue cultures continue to grow, we expect RUBY to increase in frequency and intensity.

Visual color change marks the engineering success of not only our codon-optimized RUBY sequence, but also the Gmubi promoter, AtHSP terminator, and our Plant Transformation Protocol for use in soybean tissue. Although the goal of finding a useable reporter gene took our lab through three different designs, we ultimately ended up with a reporter gene that is clearly visible to the naked eye, does not require sacrificial plant cultures, and does not require the input of potentially hazardous chemicals such as herbicides or GUS assay chemicals.

References

1. Yang, S. et al. An efficient Agrobacterium-mediated soybean transformation method using green fluorescent protein as a selectable marker.Plant Signal. Behav. 14, 1612682 (2019).
2. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic. Res. 7, 1–6 (2020).