Team:CIDEB-UANL Mexico/labwork conclusions

Capture module

After all the experiments and results interpretations, it was concluded that even if the ligation of nhaS and RFP with pSB1C3 came out with two types of bacteria (red and white), both of them had the nhaS gene functioning.

The reasons of why there were red and white colonies, first, is that the Petri dishes where the transformed bacteria were inoculated with the gene, were not covered with aluminum. This meant that the UV promotor was activated with normal light and activated the production of nhaS and RFP. The second reason is that during the purification process, the RBS and the first 50 nucleotides of the RFP region mutated because of the exposure to UV irradiation, a necessary step in the process. Because of this, bacteria transformed with the ligation of mutated fragments would not express the RFP gene, and therefore they appeared white.

The mutation of the RBS before RFP causing white bacteria and the functionality presence of nhaS in both types of colonies was proved in the sequencing and in the experiments of viability in salt.

As it was reported in the patent, the nhaS gene gave certain resistance to salt but there was not any exact percentage of the resistance. There were performed diverse experiments in order to know the maximum salt concentration bacteria transformed with the gene would survive. The concentrations where the bacteria survived were from 1% up to 15% of NaCl in the medium. It can be concluded that the bacteria survived in different NaCl concentrations up to 15%, while not transformed bacteria can survive in a 1% NaCl medium, but not in higher NaCl concentrated mediums. Another conclusion obtained from the experiments is that the bacteria with NhaS can survive in a high NaCl concnetrated medium only if it has its corresponding nutrients, because in a medium containing NaCl only it dies. We already know that the bacteria survives in a high NaCl medium, but the experiments that prove whether the bacteria captures sodium ions or not will be performed the weekend of June 21th and 22th. The results will be shown in the Jamboree presentation.

In summary, nhaS is a gene that produce a multitask protein with a lot of advantages, not only because it is pretty little and do not represent a large genetic charge to the bacteria, but also it gives many abilities to it:

●Give resistance to salty environments up to 15% of NaCl concentration

●Captures ion sodium ions

●Regulates the pH of the cell (this is the reason of the resistance it gives)

In the case of the E.CARU project, those characteristics where used with the purpose of remove sodium ions in order to desalinize water, but nhaS can be used in many different forms and in other aspects of biotechnology.

Aroma module

In the Aroma module tests, the medium with 10mM of salicylic acid was the one that produced the most intense odor. If the quantity is increased to 30m the odor is not reported. This indicates that the plasmid was actually in the bacteria and that the riboswitch worked at 35°C. In the case of the plaque that had the odor even if it was incubated at 29 ºC, it is concluded that this happened because the riboswitch is very sensitive to heat, and it was activated during the little time at which it was outside the incubator. In the case of the bacterias exposed to salicylic acid at 30mM, they presented a very similar odor despite of other conditions. This means that the 30mM concentration of salicylic acid could eliminate bateria or could affect the enzymatic reaction of the WinterGreen enzyme by saturating it.

As an extra, at first, the experiments where performed in test tubes. After obtaining an aroma result, the team did the same experiment in Petri dishes. It was appreciated that the odor was more intense in Petri dishes than in test tubes.