Developing a Framework for the Genetic Manipulation of Non-Model and Environmentally Significant Microbes
Project Results: Overview
Click on a component to learn more about our method.
The growth rate of PCC7002 was found to be highly sensitive to growth conditions, particularly light saturation and CO2 content of air. PCC7002 samples grown in CO2-supplemented conditions grew significantly faster (as measured by OD 730 nm) than samples grown in atmospheric conditions, and samples supplemented with 10mM glycerol grew to a much higher saturation than non-supplemented samples in the same period of time (glycerol supplement = top graph; no glycerol = middle graph). We also measured the ratio of chlorophyll content (OD 650 nm) to cell saturation (OD 730 nm) for cell populations grown in glycerol-supplemented versus nonsupplemented conditions. The glycerol was also found to not shunt cells away from photosynthetic pathways, as populations grown in glycerol had the same chlorophyll content per cell as nonsupplemented populations (bottom graph, PCC7002 grown in A+ medium is shown in green).
Rhizobium tropici CIAT 899 and our Sinorhizobium meliloti 1021 strains were incubated at 30C. We conducted growth assays on each strain we worked with in order to determine mid-log growth phases and doubling times; this was important in optimizing our transformation efficiency.
PCC7002 cell populations transformed with linear kanR genes flanked by 1000 bp homology arms up- and downstream of the gene demonstrated resistance to 200 µh/ml of kanamycin after a 24 hour incubation period and recovery in A+ medium. The six leftmost tubes in the picture below contain experimental samples which were recovered and inoculated in kanamycin-containing A+ medium; the four rightmost samples are all wild-type controls in kanamycin medium.
We pursued two types of transformation into rhizobia: conjugation and electroporation. While our conjugation efficiencies of the desired pKT230 plasmid into E. coli were very high (~90%), our conjugation was inconclusive in rhizobium because many of our strains had several innate antibiotic resistances and thus there was growth on the negative controls. However, once we optimized our electroporation protocols by adjusting centrifugation speeds, antibiotic concentrations, and electroporation voltages and pulses, we were able to successfully transform our plasmids of interest into rhizobia.
PCC7002 was found to be susceptible to all antibiotics tested around standard E. coli concentrations, though light-sensitive antibiotics (tetracycline and rifampicin) were less effective due to degradation in light-saturated growth chambers. Click on the links below to see the growth curves for PCC7002 samples grown in different concentrations of tested antibiotics.
Our main selection for transformation was antibiotic resistance. Before conducting electroporations, it was important to establish the innate resistances in our rhizobial strains by doing antibiotic resistance assays, both on plates and in liquid cultures. We observed that resistances changed depending on the growing media (Tryptic Soy Broth vs. LB) and conditions (liquid vs. plate). Our selection for the incorporation of LIC plasmids was sucrose.
mutS Gene Knockout
We successfully obtained mutS knockout genotypes of PCC7002 by transforming linear fragments for homologous recombination. Presumably due to the polyploidy of PCC7002 cells, however, we did not obtain homozygous ∆mutS mutants; doing so will require several more rounds of growth and plating for single colonies. Back to Top
We successfully PCR amplified promoters to express our recombinases in rhizobia as well as assembled promoter-citrine and promoter-recombinase constructs. Our band sizes were verified through gel electrophoresis.
Testing DNA Constructs
We conducted a fluorescence test in order to determine which promoters were inducible in rhizobium and what level of expression was obtainable. Before testing the promoter-citrine constructs in rhizobium, we first tested them in E. coli. In E. coli, we were able to obtain fairly high fluorescence readings for the lac and tac promoters as well as the constitutive Anderson ones. MelA and bacA are promoters specific to rhizobia, so we did not expect those to work well in E. coli. When we tested the promoters in rhizobia, we found that out of the inducible promoters, tac had the greatest expression and out of the constitutive promoters, Anderson Medium resulted in the most fluorescence. The incorporation of the promoter-citrine constructs in our organisms of interest was confirmed by sequencing.
Using search tools that compare nucleotide sequences to organisms and viruses in NCBI and UniProtKB databases, nine lambda beta and SPP1 35 homologues were identified that exist in Rhizobium species. Further reading: http://2015.igem.org/wiki/images/f/f8/Search_for_Beta_Homologue_Recombinases.pdf