Selecting Chassis

When we were designing our project, our goal was to genetically modify an organism to uptake phosphate and solubilize it to utilize unavailable phosphate and prevent unwanted runoff. Originally, we wanted to engineer a plant to be able to solubilize phosphate, but we were worried that engineering a eukaryotic organism would not fit the scope of this project. However, we wanted to prioritize the use of plants since our team is based in two coastal cities at risk of flooding due to the lack of greenery since encouraging our city governments and people to plant more greenery will help protect our communities from flooding. While researching, we encountered the bacteria rhizobia, which has a symbiotic relationship with plants, solubilizing phosphate so that it can be available to plants. Because of this pre-existing relationship, we decided to engineer rhizobia to increase its uptake and conversion of phosphate. This allowed us to engineer a prokaryotic organism, which would fit the scope of our project, while also encouraging the use of plants in our coastal cities.

Choosing Soil Media

When researching prior projects and experiments related to Rhizobium tropici, we encountered three different types of media in which the bacteria were grown by the (2019 Tacoma team). In order to determine which media we should use for our experiments, we conducted a trial in each media. The optical densities of the different samples at different times are recorded in the table above as well as in the graph below. A higher optical density indicates that there is a higher concentration, and therefore quantity, of bacteria. The negative readings for samples 3 and 4 are likely machinery errors due to high concentrations of Ca causing opacity, and due to those readings it was not possible to generate an equation for the OD in the soil media. Overall the yeast mannitol with low calcium showed an exponential growth of y= 0.0479e0.445x with an r2 value of 0.99. The yeast mannitol with high calcium showed an exponential growth of y=0.0343e0.4582x with an r2 value of 0.94. A t-test paired two samples for means performed between the ATCC 111 media, which was the most commonly used one in literature, and the yeast mannitol extract with low Ca concentrations yielded a p-value of 0.11 (greater than the threshold of 0.05) showing no significant statistical difference found in growth. A t-test of two samples for the means between the low Ca concentration yeast mannitol and high Ca concentration yeast mannitol yielded a p-value of 0.11. Therefore, since there was no statistical difference in growth, it was decided to use the yeast mannitol low Ca soil media since a low Ca was preferred for increased accuracy of measurements and lower cost and there was no evidence to suggest that the yeast mannitol with higher concentrations of Ca would yield higher rhizobium ODs.

Design of Plasmid

Our research and guidance from our mentors led us to discover two key pathways in Rhizobacteria for mineral phosphate solubilization: the Pqq(FABCDEG) genes, which are believed to play a role in Pyrroloquinoline quinone (Pqq) biosynthesis as a redox cofactor to glucose dehydrogenase (GDH) enzyme, and the pst(SCAB) genes, which have been identified as being involved in phosphate transportation. We chose to use the pBBR1MCS vector in the pBBR1MCS-2 plasmid from addgene ( that the 2019 Tacoma team used because this vector contained the lac operon, a well documented promoter, and it contains a multiple cloning site with blue white selection which would allow us to easily see if our plasmid was transforming successfully. Since this lac promoter is a derepressed promoter in E. coli, we chose it because it may be constitutive in R. tropici.

We decided to use these chosen genes in a coupled approach to solving the issue of phosphate contamination: solubilizing phosphate to make it available to plants (with the Pqq and GDH) and to increase the uptake of the mineral phosphates (with the pst(SCAB) transporter) by rhizobia. We chose the lac operon promoter for our codon optimized genes because we desired constitutive promoter for our construct and the lac operon promoter is both constitutive and very well-documented. We codon optimized these genes with Benchling and had them synthesized by Twist Bioscience.

Build Plasmid

To figure out how best to transform our plasmid into rhizobia, we practiced transforming the plasmid into rhizobia before we had cloned our genes into the plasmid. For our first attempt, we followed a blue-white screening procedure from the 2014 Hannover team for rhizobium electroporation. To test that our plasmid was functional, we performed a blue-white screening. The intact plasmid contains a functioning LacZ gene, so when we add Xgal and IPTG to the plasmid, it should turn blue. However, when the genes have been inserted into the plasmid and the LacZ gene has been disrupted, the plasmid should turn white.

We performed the blue-white screening on the plasmid before cloning our genes to ensure that the plasmid was operational. The results of this procedure were not successful; no bacterial colonies turned blue and mold had formed on the plates, compromising the validity of the results. In our next attempt, we changed the concentration of DNA while following the same procedure and holding all other variables constant and ensured that the lids of the petri dishes were tighter than before to prevent any mold from getting in and disturbing the samples. This time the results showed blue colonies, showing that our plasmids had an operating LacZ gene.

Results of Cloning

The plasmid that we are using for cloning, pBBR1MCS-2, has a LacZ gene in it, so we can do blue-white screening in E. coli to see if our genes were inserted. Our blue-white screening showed that our plasmids were successfully assembled in E. coli. From there, we screened the plasmids by PCR, isolated the plasmids from E. coli, and then transformed them into Rhizobia. We then screened our Rhizobium by PCR, and our PCR and gel electrophoresis experiments showed that our transference to rhizobia was successful. We were able to confirm the length of inserted fragments. We expected a fragment length of around 3700 base pairs for pstSCAB and a fragment length of around 3200 for PqqC + gcd. Colonies that begin with the numbers 1 & 2 contain pstSCAB (~3700), and colonies that begin with the numbers 3 & 4 contain PqqC+gcd (~3200) From the image below, samples 1-1, 1-2, and 2-1 showed successful gene insertion of pstSCAB genes; samples 3-3, 3-4, 4-2, 4-3, 4-4, 4-5, and 4-6 showed successful gene insertion of PqqC+gcd genes.

We then transformed our plasmid into e.coli. Once we had successfully transformed them into e.coli, we transformed them into rhizobium. Our PCR test showed that our Rhizobium contained our genes, but we wanted to see if the proteins were actually produced from the inserted genes. Our first protein gel did not provide conclusive results. The ladder we used, (SMOBIO ExcelBand™ 3-color Regular Range Protein Marker, PM2500), allowed us to see that most of our proteins were under 25000 Da, which is not expected because the masses of our proteins are greater than 25000Da. In addition, there appeared to be a lot of debris in the gel-- either from cell lysis or proteins congregating. We ran another protein gel, ensuring cell debris is not inside the gel. This yielded more success as we saw promising results for gcd protein synthesis. We expected 87 kDa for GDH enzyme and saw a corresponding band for one of our PqqC+gcd samples- the arrow marks the band in the image below. It is worth noting that we were expecting to see the proteins running at their individual sizes, but because pstA, pstB, and pstC form the pstABC transporter and, therefore, form strong associations, we were also aware that we might see them still associating in the gel and a protein complex at a larger size. This would correspond to around 95 kDa.

For the future, we will continue phosphate uptake and solubilization assays.