Difference between revisions of "Part:BBa K2055369"

Latest revision as of 19:22, 30 October 2016

Na(+)/H(+) antiporter NhaA generator

This construct codes for intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli capable of excreting one intracellular Na(+) ion in exchange for two H(+) external protons, by doing this the pH within the cell is maintained at a constant level. This protein is active at alkaline pH. This construct is regulated by a weak constitutive promoter and its objective is to augment bacterial resistance to alkaline pH.

We transformed E. coli BL21 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 415
Illegal BamHI site found at 1120
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 382
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K2055369 short</partinfo>
-This construct codes for intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli capable of excreting one intracellular Na(+) ion in exchange for two H(+) external protons. This protein is active at alkaline pH. This construct is regulated by weak constitutive promoter and its objective is to augment bacterial resistance to alkaline pH.
+This construct codes for intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli capable of excreting one intracellular Na(+) ion in exchange for two H(+) external protons, by doing this the pH within the cell is maintained at a constant level. This protein is active at alkaline pH. This construct is regulated by a weak constitutive promoter and its objective is to augment bacterial resistance to alkaline pH.
 https://static.igem.org/mediawiki/parts/thumb/c/c7/T--Tec-Monterrey--protonpump-final.png/800px-T--Tec-Monterrey--protonpump-final.png
-We transformed E. coli BL21 with three different constructs, one coding for the proton pump, and the other two for GolS and TetH, which were used as control parameters to compare their growth rate at a constant alkaline pH adjusted with KOH. We measured OD600  of three replicates in approximate intervals of 60 minutes for 8 hours. 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. As it is shown in the graphic, the strain transformed with the proton pump had an exponential growth, while the other two couldn’t resist the alkaline pH and had minimal growth. This modification to bacterial cell membrane may increase stress resistance under alkaline conditions
+We transformed E. coli BL21 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).
-(by maintaining the pH within the cell at a constant level, this is done by the entrance of two protons and exit of a sodium cation.)
+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.