Results!

To date, we have successfully tested a promoter (BBa_K4201000) and a terminator (BBa_K4201009) for use in soybeans. We have repeated three cycles of the engineering process to determine a suitable reporter gene (BBa_K4201010, BBa_K4201011, BBa_K4201012). Final vectors pLSUK TDS2_CrtE_RUBY and pLSUK Crte-cytoTDS-MBP_RUBY have had their assemblies confirmed by sequencing (Figure 1) and plsuk crte-TDS is undergoing active testing in soybean cultures.

The reporter that works best in our engineered system is RUBY. Because RUBY is present in our transgenic construct, visible expression of RUBY indicates that our transgene has been integrated into the genome of the soybean in that location. GC-MS of these RUBY regions can tell us the efficiency of our engineered construct by detecting the levels of Taxadiene, and more specifically the levels of the two isomers produced by Taxadiene Synthase.

An unfortunate failure we had to contend with was the time allotted to reporter genes that we could not end up using. We spent a significant part of the project building parts with GUS and GFP before moving on to RUBY. Because of this, we were not able to get our Crte-cytoTDS-MBP_RUBY and Crte-cytoTDS2-MBP_RUBY constructs into soybeans in time for them to grow before the wiki-freeze. We were hoping to have some GC-MS data, but we had to reassemble our constructs with RUBY. Despite the setbacks, we now have a reliable reporter to use to continue our research. One thing to consider in the future with RUBY is its size(~5000bp); it is significantly larger than GFP (~700bp) or GUS(~2500bp). This could become a problem when we start to encode more of the paclitaxol synthesis pathway in our construct.

One of our composite parts, Crte-cytoTDS-MBP_RUBY, is currently transformed into 40 soybean tissue cultures and should allow the soybeans to begin producing the first two intermediates of the paclitaxel pathway. Out of the 40 soybeans, 36 were still viable after being introduced to agrobacterium. We are currently able to see visible signs of our reporter gene in these tissue cultures (Figure 2). Prior to the Jamboree, we should have quantitative data from GC-MS on this composite part compared to non-transformed soybeans. To measure this we will send off samples to the Guangxi-U-iGEM Team from the beans transformed with Crte-cytoTDS-MBP_RUBY. The samples will be extracted from the spots expressing visible RUBY and samples on the same plants without visible RUBY. In addition, we will test plants with only RUBY and no taxadiene synthase. This will allow us to compare betalain intensity to GGPP and taxadiene intensity, as well as understand how taxadiene may be transported across cell membranes because it is volatile. The control would allow us to confirm that our peaks would actually be a new product. GC-MS will allow us to differentiate between the two isomers produced by TDS.

We are in the process of constructing many other composite parts for further testing. The next steps after achieving successful biosynthesis of Taxadiene would be the integration of T5aOH and TAT into our constructs. These enzymes are the subsequent steps in the paclitaxel biosynthesis pathway and the addition of these enzymes would bring us closer to our engineering goal of synthesizing Baccatin III.

In addition to the biosynthesis of paclitaxel, we are introducing a new biosafe chassis to iGEM, soybeans. Soybeans are a safe chassis to work with because they have a minimum separation distance of 6 feet due to heavy pollen¹. Our goal is to make this chassis safer by engineering the flowers to be cleistogamous. This will ensure that the genetically modified pollen will remain inside the flower and never get released into the surrounding environment. We are in the process of designing this construct and would like to implement it in future iGEM Jamborees.

Testing these plasmids in preparation for next year's iGEM will tell us what constructs that we have designed are promising and which ones are not. This will allow us to pinpoint what constructs produce the highest levels of our desired taxadiene isomer, so we can add the subsequent enzymes in the paclitaxel biosynthesis pathway in order to get closer to our engineering goal.

Results!

References