Microorganism Isolation and Characterization
We successfully isolated and purified uncharacterized Myxobacteria from Chihuahua’s soil. When exposed to stress factors (such as starvation), Myxobacteria tend to form fruiting bodies. These amazing microorganisms are able to secrete almost 300 different metabolites with different uses. Even though they have been studied, they have not been used for any iGEM project until now!
The inhibitory activity of our isolated Myxobacteria was proven by confronting them with previously isolated fungus.
However, our project needed our Myxobacteria to be confronted with fungus isolated from crops. Our main objective was to test its action against R. solani, and see if Myxobacteria had inhibitory action against it. Confrontations were performed in PDA agar plates, Myxococcus xanthus against Rhizoctonia solani and later against Rhizopus oryzae. It was determined that unmodified Myxobacteria are able to inhibit fungal growth. A major inhibition was observed in Rhizoctonia than in Rhizopus. Other confrontations were performed against uncharacterized fungi only to challenge Myxobacteria.
In our visits to the crop fields, we collected some samples of infected alfalfa. We wanted to know for sure what was the reason of the infection and what part of the plant its attacked. The team found some fungi around the root and took them back to the lab to try to characterize them. We were able to successfully characterize a fungus macro and microscopically, suspected to be R. oryzae.
The first attempt to build our BioBrick™ consisted in the assembly of eight different parts. We transformed E. coli with each of our BioBricks™ to increase the quantity of DNA we had, then we extracted the plasmid of each one of them so we could make the assembly. Each plasmid that was extracted was ran in an electrophoresis gel so we could be certain that what we obtained was what we wanted.
When we did the last electrophoresis we had as a result that all the parts that we extracted were right, with the exception of one of them, we believe that the chloramphenicol backbone was supercoiled and that’s the reason it appeared to be at a higher molecular weight.
After this we quantified how much nanograms of DNA we had per microliter and proceeded to do the 3A assembly. Since not everything is digested and ligated properly during the assembly, there are some backbones without the insert that can interfere. Because of this there were two types of colonies in the plates where our transformed cells were plated: pink and white. The ones that we were interested in were the white ones, since these are the ones that have what we want correctly inserted; the pink ones are the backbones that were not correctly digested and ligated and thus are of no use for us. We grab one of the white colonies and put it to grow in LB broth, so we could later extract its plasmid and start this process again until we have the BioBrick™ we desire.
The BioBrick’s we built contains intermediate part of CDS of the chaperonin 60 (along with chaperonin 10 provides resistance to high temperatures) and CspC (it provides resistance to low temperatures), BBa_K2069000: INT 3. Also, another that allows the easy assembly for expression of a cephalopod Reflectin 1A with a T7 promoter, BBa_K2069001: INT 1. The last one give expression of Reflectin 1A and GAFP-1 under control of a T7 promoter, BBa_K2069007: Eme.