Original Iteration of our Project/Modifications Made and Why
Originally we wanted to modify rhizobia to allow it to form a symbiotic relationship with other plants. However, in the course of our research, we found that rhizobia is not currently as widely used as we had expected. A number of inadequacies are present in wild-type rhizobia, rendering them impractical for wide-spread use in commercial agriculture [1,2]. Additionally, we found that legumes have an untapped potential for improving agricultural sustainability  and economic stability in developing countries, as they require little to no fertilizer  and are ideal for soil revitalization . Thus, we put our plans of spreading rhizobia to other types of crops on hold while we first worked to improve rhizobia for legumes.
A highlight of our research was the 2008 paper “Improvement of Drought Tolerance and Grain Yield in Common Bean by Overexpressing Trehalose-6-Phosphate Synthase in Rhizobia” by Suarez et. al from Autonomous University of the State of Morelos. This publication described the engineering of a strain of rhizobia that increased the crop yield, nitrogen fixation, and stress tolerance of inoculated legumes. 
Our first step to the development of this improved rhizobia hub was to verify the findings made by Suarez et. al. This meant replicating their research as closely as possible. Unfortunately, due to the resources available, we had to make some key changes. The vector used in their research, pBBR1MCS-5, was not something that our team could purchase. A sister vector, pBBR1MCS-2, is available through Addgene and features very few modifications . We decided to use this plasmid to follow the original publication as closely as possible.
The otsA gene was derived from the complete genome of Rhizobium etli (NC_007761.1) and confirmed using protein alignment against trehalose-6-phosphate synthase (Q66Q98). To make this sequence compatible with BioBricks, we made 4 silent mutations to remove PstI sites and 1 silent mutation to remove an EcoRI site. The original sequence we derived had a high GC content that was likely to cause complications during synthesis. We made 6 additional silent mutations to decrease GC content.
Our final successful cloning pathway was digesting the otsA gene and pBBR1MCS-2 with KpnI/HindIII, ligating, then transforming into DH5Alpha. We then miniprepped to extract the plasmid, verified the sequence, and then electroporated it into R. etli.
Attempt to Redesign Cloning Pathway
Due to a clerical error, our Twist order was not processed when we intended. As a result, we had a significant delay in cloning. In an attempt to decrease time spent waiting for delivery, we ordered the otsA gene in two parts from IDT: ots1 and ots2. To make the complete otsA gene, we first digested ots1 and ots2 with NdeI, ligated them together, then verified the full-length gene with PCR using a primer set that binds to the BioBrick sites.
After obtaining this otsA gene, we attempted to clone it in to pBBR1MCS-2 as planned originally (KpnI/HindIII digest). After screening numerous colonies with no positive results, we changed our approach. The lack of clones could have been due to the poor insert quality, so we amplified the gene using the BioBrick primers, then proceeded through cloning using EcoRI/SpeI. Again, no colonies contained the correct sequence. These four start-to-finish attempts at cloning pBBR1MCS-2/otsA using the ligated otsA gene were, ultimately, fruitless.
Our Plans to Test
We will be performing tests on:
- Overexpression Plasmid in R. leguminosarum
- Desiccation Tolerance
- Freeze Thaw Tolerance
- Nodulation Frequency
- Salinity Tolerance
- Plants Inoculated with Ox Strain of R. leguminosarum
- Crop Yield
- Drought Tolerance
These tests will be the basis of our research but will simply be building blocks for our own individual research. After the completion of these preliminary tests with the R. etli strain we intend to repeat them again using various other strains, beginning with R. leguminosarum. Due to strain availability, we have yet to receive R. etli and are instead beginning our research with R. leguminosarum.
- 1 - Graham, P. and Vance, C. (2003). Legumes: Importance and Constraints to Greater Use. Plant Physiology, 131(3), pp.872-877.
- 2 - Herder, Griet Den, and Martin Parniske. “The Unbearable Naivety of Legumes in Symbiosis.” Current Opinion in Plant Biology, vol. 12, no. 4, 2009, pp. 491–499., doi:10.1016/j.pbi.2009.05.010.
- 3 - Chianu, Jonas. N., et al. “Biological Nitrogen Fixation and Socioeconomic Factors for Legume Production in Sub-Saharan Africa: a Review.” Agronomy for Sustainable Development, vol. 31, no. 1, 2011, pp. 139–154., doi:10.1051/agro/2010004.
- 4 - Smith, M. Scott, et al. “Legume Winter Cover Crops.” Advances in Soil Science, 1987, pp. 95–139., doi:10.1007/978-1-4612-4790-6_3.
- 5 - Suarez R, Wong A, Ramirez M, et al. Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia. Mol Plant Microbe Interact. 2008;21(7):958-966. doi:10.1094/MPMI-21-7-0958.
- 6 - Kovach, Michael E., et al. “Four New Derivatives of the Broad-Host-Range Cloning Vector pBBR1MCS, Carrying Different Antibiotic-Resistance Cassettes.” Gene, vol. 166, no. 1, 1995, pp. 175–176., doi:10.1016/0378-1119(95)00584-1.