Team:Tec-Monterrey/Results
Module 1
CVR usually measures the composition in order to sell their slag to other companies for further use. For the digestion, the particle size was 1-2 mm. To analyze its content of metals, 10 mL of aqua regia was added to a flask containing 1 gram of slag. It was left for 12 hours and then the solid residues were removed by filtration. The liquid sample was then analyzed and the metal concentrations (mg/kg) were obtained, as well as the composition percentage.
We measured the composition of the slag with the technique Atomic absorption spectroscopy (AAS), Juan José Cortés helped us with the procedure and we obtained these results.
The weight of the dry slag after the digestion was 0.327 grams, therefore, the total grams of metal per gram of slag must be of 0.673, there are 0.0219 grams of metals left to calculate the composition so we asked Julio Rivera to conduct an ICP-OES to calculate them and we obtained these results.
Therefore for our model, we know what is the maximum solubilization of metals.
Module 2
Heat shock transformation of Chromobacterium violaceum with the RFP-generator (BBa_J04450)
We successfully transformed a novel chassis, Chromobacterium violaceum. Although transformation using an electroporation protocol had been described before, there was no previous report of a heat shock transformation of C. violaceum. We adapted standardized E. coli protocols for calcium competency and heat shock transformation, successfully transforming C. violaceum.
As seen in the figure, we tried preparing the calcium competent cells at different optical densities and transformed using different durations of heat shock. We obtained the best efficiency with competent cells prepared with an O.D. 600 of 0.5 and 60 seconds of heat shock at 60ºC.
We also showed that the part BBa_J04450 can be used as a reporter gene in a novel chassis, Chromobacterium violaceum. This was necessary because, as C. violaceum produces a native purple pigment (violacein), we were curious as to whether RFP would be useful as a reporter gene. We reasoned that it could be possible as violacein production is regulated by quorum sensing and, therefore, only produced after cell population density increases considerably (in our experience, typically more than 24 hours after plating).
Additionally, we used the RFP-generator (BBa_J04450) in pSB1C3 in the transformation. We found no previous report in the literature of the use of pSB1C3 in C. violaceum, so we proved that it can indeed be used in this new chassis, with the advantages of introducing a new chassis to iGEM that can be transformed using the standard plasmid.
Characterization of BBa_R0010 in Chromobacterium violaceum
We characterized the promoter BBa_R0010 in a novel chassis, C. violaceum. BBa_R0010 is lacI regulated, however, we were unable to confirm whether or not C. violaceum produces lacI natively through a literature search, and BLAST analysis of its genome suggested that it does not have this regulating system. Thus, we compared the levels of RFP expression with and without IPTG induction. There were no significant difference in the fluorescence of the between the induced and uninduced groups of C. violaceum, whereas in E. coli it showed normal induction and a clear difference of RFP expression when the inductor was added to the bacteria. Therefore we can confirm that this promoter works differently in C. violaceum than in E. coli. Our conclusion is that the promoter BBa_R0010 can be used as a constitutive promoter in C. violaceum.
Characterization of a new part:
BBa_K2055369 (Na(+)/H(+) antiporter NhaA generator)
We characterized a new part: BBa_K2055369. This part is a proton pump generator that is regulated by a weak constitutive promoter from the Anderson collection.
The purpose of these biobrick is to improve the survival under the alkaline conditions that will be found in our reactor (pH 9.0-9.5). With the work of E. Padan et al. (2005), we found that monovalent antiporters play an important role in alkaline pH homeostasis, in addition to roles in Na+ and volume homeostasis. Similarly, Na+/H+ antiporters play essential roles in homeostasis of pH, Na+ and volume in bacteria. Thus, we reasoned that addition of a proton pump could improve survival under basic conditions by increasing proton transport across the membrane. Specifically, we used the intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli. It excretes one Na(+) ion in exchange for two H(+) external protons. This protein is active at alkaline pH.
We transformed BL21 E. coli with three different constructs, one coding for the proton pump, and, as control groups, two constructs that consisted of the same plasmid as our proton pump (pUC-IDT, which has ampicillin resistance), but without the actual coding sequence for the proton pump. However, in previous experiments we noticed that one of these two constructs actually reduced the viability of the bacteria, thus we determined it was not a reliable control group for our experiment. The other construct, which we report as our control, had no difference in growth compared to our proton pump under non-modified pH conditions (regular LB media). For our experiments, we compared the growth rate at a constant alkaline pH of 9.3, that was manually adjusted with KOH, at a growth temperature of 37ºC and 250 rpm. We conducted our experiments in triplicate, measuring the OD600 of each of them in approximate intervals of 60 minutes for 8 hours starting at an OD of 0.1. We calculated an average of the three replicates for each transformed strain and graphed the data in order to determine if the proton pump had a significant effect on the survivability of the bacteria. The strain transformed with the proton pump had an exponential growth phase, while the others couldn’t resist the alkaline pH and had minimal growth. This modification to bacterial cell membrane may increase stress resistance under alkaline conditions.
Characterization of the golS promoter in Chromobacterium violaceum
We submitted a new part: BBa_K2055369. As the pgolS promoter has a basal transcription level, some CueR would be constantly expressed. CueR is a transcription factor that stimulates the transcription at the pgolS promoter when bound to copper or gold, as was described by the iGEM York 2013. Thus, this construct increases its own expression through positive feedback in the presence of gold or copper through the production of a copper/gold sensitive protein. We inserted a Red Fluorescent Protein (BBa_K51603) after this promoter and tested its efficiency in C. Violaceum. In C.Violaceum, this part expressing the RFP, did not presented an inducible activity in the e-waste copper and gold compositions that Tec-Monterrey 2016 worked with. Nevertheless, a basal activity for the fluorescence was found in the following graph
We used 2 different concentrations of copper as well as 4 different compositions of the shredded e-waste that CVR provided.
ReferencesPadan, E., Bibi, E., Ito, M., & Krulwich, T. A. (2005). Alkaline pH homeostasis in bacteria: New insights. Biochimica et Biophysica Acta - Biomembranes, 1717(2), 67–88. http://doi.org/10.1016/j.bbamem.2005.09.010
Brought to you by iGEM Tec-Monterrey 2016
