Team:CIDEB-UANL Mexico/labwork conclusions

From 2014hs.igem.org

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<p>From the results obtained we can conclude that:</p>
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<p><b>Capture module</b></p>
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<p>1) The NhaS module gave the bacteria the ability to survive in a medium with high NaCl concentrations. The concentrations where the bacteria survived were from 1% up to 15% of NaCl in the medium. Some bacteria appeared to be red indicating that the NhaS gen is active, but some of them appeared to be white although they survived in the medium, indicating too that the NhaS gene was activated. The reason that appeared red and white colonies was due to a mutation in the RBS region or at the beginning of the RFP region as it was interpretated in the sequenciation of both samples. It can be concluded that the bacteria survived in different NaCl concentrations up to 15%, not transformed bacteria can survive in a 1% NaCl medium, but not in higher NaCl concentrated mediums. It was also found that the NhaS gives the bacteria resistance to a salty medium even though that it has not the IrrE gen. 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 with only NaCl 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.</p>
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<p>After all the experiments and interpretations of results, it be concluded that even if the ligation of nhaS+RFP with pSB1C3 gave two types of bacteria (red and white), both of them had the nhaS gene functioning. The reasons of why there was red and white colonies, first, is that the Petri dish where were inoculated the transformed bacteria with the gene, was not covered by aluminum, so 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 RPF region mutated because of the exposure to UV irradiation, a necessary step in the process. Then, bacteria transformed with the ligation of mutated fragments, would not express the RFP gene, therefore they were white.</p>
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<p>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.</p>
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<p>As it was reported that the gen nhaS  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 with only NaCl 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.</p>
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<p>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:</p>
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<p> Give resistance to salty environments up to 15% of NaCl concentration</p>
 +
<p> Captures ion sodium ions</p>
 +
<p> Regulates the pH of the cell (this is the reason of the resistance it gives)</p>
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<p>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.</p>
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<p>2) In the Aroma module tests, the medium with 10mM of salicylic acid was the one that produced the most intense odor and 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 actually worked at 35°C.  In the case of the plaque that had the odor even if it was incubate 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 winter green enzyme by saturating it. </p>
<p>2) In the Aroma module tests, the medium with 10mM of salicylic acid was the one that produced the most intense odor and 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 actually worked at 35°C.  In the case of the plaque that had the odor even if it was incubate 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 winter green enzyme by saturating it. </p>

Revision as of 01:21, 21 June 2014

iGEM CIDEB 2014 - Project

Conclusions

Capture module

After all the experiments and interpretations of results, it be concluded that even if the ligation of nhaS+RFP with pSB1C3 gave two types of bacteria (red and white), both of them had the nhaS gene functioning. The reasons of why there was red and white colonies, first, is that the Petri dish where were inoculated the transformed bacteria with the gene, was not covered by aluminum, so 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 RPF region mutated because of the exposure to UV irradiation, a necessary step in the process. Then, bacteria transformed with the ligation of mutated fragments, would not express the RFP gene, therefore they were 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 that the gen nhaS 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 with only NaCl 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.

2) In the Aroma module tests, the medium with 10mM of salicylic acid was the one that produced the most intense odor and 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 actually worked at 35°C. In the case of the plaque that had the odor even if it was incubate 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 winter green enzyme by saturating it.

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