Soil Media
When researching prior projects and experiments related to Rhizobium, we encountered three different types of media in which the bacteria were grown. We obtained our strain from ATCC, which recommended ATCC#111 media, which contains African violet soil in it. This was a difficult media to make, so we wanted to see if we could instead use yeast mannitol media, which we found in the literature to work with R. tropici (del Cerro P, Rolla-Santos AA, Gomes DF, Marks BB, del Rosario Espuny M, Rodríguez-Carvajal MÁ, Soria-Díaz ME, Nakatani AS, Hungria M, Ollero FJ, Megías M. Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899. BMC Genomics. 2015 Oct 26;16:864. doi: 10.1186/s12864-015-2033-z. PMID: 26502986; PMCID: PMC4624370.) The mannitol media had a high concentration of calcium and was cloudy, which made it hard to use to measure bacterial growth. So, we also tried lowering the calcium concentration to its solubility level in yeast mannitol media (15 mM).
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 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 errors due to high concentrations of Ca which made the values of our blanks very high. 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. Therefore, since there was no statistical difference in growth; it was decided to use the low Ca yeast mannitol media since a low Ca was preferred for increased accuracy of measurements and there was no evidence to suggest that the yeast mannitol with higher concentrations of Ca would yield higher rhizobium ODs.
Bacterial Transformation
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.
Phosphate Assays
To determine whether inserting the pstSCAB and PqqC+gcd genes increase phosphate uptake from the media, we looked that the cells’ ability to uptake phosphate (making halos) from plates with high levels of tricalcium phosphate. We tried various bacterial dilutions with varying bacterial spot amounts. We used R. tropici as a negative control, so no results were expected. We used P. fluorescens as a positive control as literature indicated it had phosphate uptake abilities (K.‐H. Park, C.‐Y. Lee, H.‐J. Son, Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth‐promoting activities, Letters in Applied Microbiology, Volume 49, Issue 2, 1 August 2009, Pages 222–228, https://doi.org/10.1111/j.1472-765X.2009.02642.x). However, P. fluorescens is highly motile. We concluded that the agar plates were too "wet" to achieve good results, and the P. fluorescens appeared to move across the entirety of the plate, making it nearly impossible to detect phosphate uptake if any occurred. So, on October 1, we used the laminar flow hood to remove excess liquid. Then, we proceeded with 9 assay plates with the samples listed in the table below. After 7 days of incubation, the bacteria grew well and appeared concentrated enough to provide clear results. However, no halo was visible for any of the samples, proving our experiment did not work as planned. We believe this is due to the media we used (low calcium yeast mannitol) as the literature from which we derived the experiment used different media with tryptophan. For future experiments, we would like to try these plate assays again with the media used in the literature and see if we can produce similar results. (plate images in order from 1-9; 1L & 2L contain PqqC+gcd and 4L contains pstSCAB)
Protein Gels
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.
Plant Experiments
Our ultimate goal is to measure the micro symbiosis between our Rhizobium and our various plant species (Common Bean, Soybean, Showy Pink Trefoil, American Senna.). We picked the common bean and soybean because these are plants that are know to participate in symbiosis with Rhizobium. We also wanted to know if we could implement the project using native plants as our micro symbionts. So we picked showy pink trefoil because they can grow in almost any soil, are indifferent to soil acidity, and germinate in 2-3 weeks. We also picked American Senna because it grows in both New York and Maryland, is a perennial whose normal life span exceeds 2 years, it has special value to bumble bees, it grows well in dry to moist soil, and it grows well in sunshine to partial shade.
We are also trying to measure if our Rhizobium is actually beneficial for the plant's growth or if it experiences phosphate poisoning when we cause it to uptake more phosphate. If we find negligible differences in growth over time, or if we see no differences, then we will know that either the Rhizobium never took root or that the Rhizobium doesn’t help the plant that much. We need to do this in order to see if our “built in killswitch” theory holds true that the plants would die from picking up too much phosphate and could then be reused as a source of phosphate themselves. In the beginning, we planned a series of experiments to test the optimum conditions for our Rhizobium to survive. This included assessing what the best plant age for inoculating is. Unfortunately, our first Rhizobium check proved fruitless aside from a single set of nodules on a showy pink trefoil.
We are not sure why this is happening, but we looked over other protocols and saw that most experiments used a greenhouse, which we do not have. For the past week or two we have been studying a hypothesis that it may be the lower temperature that caused our lack of success since we are growing our plants at room temperature. As a result, we are now incubating our plants at 29° C. We will then contrast the growth rates with our previous rates to see if the warmer temperatures help nodules to form as expected.
Future Plans
With more time, we would conduct replicates of all our experiments to get the most reliable results. Rather than using the plate assays for the phosphate assays, we instead are using a Phosphate Colorimetric Kit (Sigma MAK030) to measure phosphate levels with a color-based assay. Additional experiments we would conduct include inoculating plants with our modified rhizobia containing the genes, experiments varying the cfu count when inoculating plants with rhizobia to determine the optimal cfu count for rhizobia inoculation in different species, and experiments measuring inoculation in different pHs and temperatures to determine the optimal conditions for rhizobia nodulation. We would perform these future experiments in a growth room where the plants would have more space and we could more easily control temperature, humidity, and light schedule to yield more accurate results.
Analysis of Results
Although many of our experiments did not go as planned, we learned from our mistakes and plan to use these lessons to successfully perform future experiments. We saw early successes in determining an ideal growth media for our bacteria and for our experimental purposes. When we began transformation, we encountered some issues with gene insertion. Upon review, we found an error in our gene constructs-- there was an issue with using restriction digest to make our recombinant DNA because we had an extra restriction site inside our gene. So, we modified our approach and used PCR to amplify the portion of the plasmid that did not include the multiple cloning site, and we used HiFi DNA assembly to integrate our genes. This led to successful creation of recombinant DNA and, therefore, allowed us to insert our genes into R. tropici and successfully clone our bacteria. Upon colony PCR, we validated that numerous colonies had successfully accepted our genes. However, when we performed phosphate plate assays and a protein gel to confirm the production and functioning of our genes, we encountered unexpected results. The plate assays took 7 days to measure due to the incubation period. This meant we were unable to foresee issues, such as the wet plates. Therefore, over the course of a month, we were unable to produce viable results. However, we experimented a lot with different bacterial solutions, bacterial spot amounts, and steps to remove extra liquid on the plates. So, we have knowledge that we can base our future experiments on with the hopes of producing viable results.
Project Achievements
Successful
- We found the doubling time of rhizobia in different medias
- We successfully ligated the pqq and gcd DNA into our plasmid and confirmed this with PCR and gel electrophoresis
- We successfully transformed our plasmid into E.coli
- We successfully transferred the plasmid from E.coli into rhizobium
Unsuccessful
- We could not interpret our phosphate assays
- We have not yet gained results of colorimetric phosphate assays and the protein gels
- We were not able to observe consistent nodulation of rhizobia in our plant experiments