Difference between revisions of "Part:BBa K2230022"

Latest revision as of 00:09, 1 November 2017

STM1128/pSB1C3

Salmonella typhimurium LT2 has two glucose-specific transporter systems, PTS system and sodium/glucose cotransporter. PTS system contains two subunits IIA encoded by crr and IIBC by ptsG which are assembled to a high-affinity active transporter. The other is a Na+/glucose cotransporter encoded by STM1128 that contributes to facilitated transport with lower glucose affinity.

Research

Based on our research, the glucose transporter of Salmonella has a lower Km compared to human small intestine, Staphylococcus and E. coli, indicating a higher efficiency for glucose uptake. In our study, we demonstrated glucose absorption ability by overexpressing each of these two systems from Salmonella in E. coli. Please go to our wiki page ([http://2017.igem.org/Team:Mingdao/Demonstrate#demonstrate-md Mingdao iGEM 2017]) for more information.

Cloning

The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3. This part has been sequenced.

Demonstration for Glucose Absorption

In order to express the genes, we chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280), was used and assembled with the transporter genes.

To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34ug/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.

Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.

Fig. 3 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was greater in Na+/Glu group than in control group with 1.2 times difference.

In our results, glucose absorption by Na+/glucose transporter expressing bacteria was achieved to 97% after 4 hours with 1.2 times enhanced efficiency compared to the normal E. coli. To further increase the rate of glucose uptake, one may think about the glucose metabolism or conversion to other materials when entering into the cell.

Reference

1. Glucose Galactose Malabsorption. American Journal of Physiology - Gastrointestinal and Liver Physiology 1998;275:G879-G882

2. Functional Properties and Genomics of Glucose Transporters. Curr Genomics. 2007;8(2): 113–128.

3. The SLC2 (GLUT) Family of Membrane Transporters. Mol Aspects Med. 2013;34(0): 121–138.

4. Glucose and Glycolysis Are Required for the Successful Infection of Macrophages and Mice by Salmonella enterica Serovar Typhimurium. Infect Immun. 2009;77(7): 3117–3126.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1255
1000
COMPATIBLE WITH RFC[1000]

@@ Line 10: / Line 10: @@
 === Research ===
-Based on our research, the glucose transporter of Salmonella has a lower Km compared to human small intestine, Staphylococcus and E. coli, indicating a higher efficiency for glucose uptake.
+Based on our research, the glucose transporter of ''Salmonella'' has a lower Km compared to human small intestine, ''Staphylococcus'' and ''E. coli'', indicating a higher efficiency for glucose uptake. In our study, we demonstrated glucose absorption ability by overexpressing each of these two systems from ''Salmonella'' in ''E. coli''. Please go to our wiki page ([http://2017.igem.org/Team:Mingdao/Demonstrate#demonstrate-md Mingdao iGEM 2017]) for more information.
-[[File:Mingdaophil1026-2.png|700px]]
+[[File:Mingdaophil1026-2.png|550px|center]]
@@ Line 19: / Line 19: @@
 The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3. This part has been sequenced.
-[[File:Mingdaophil1026-3.jpeg|400px]]
+[[File:Mingdaophil1026-3.jpeg|400px|center]]
@@ Line 26: / Line 26: @@
 In order to express the genes, we chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp. The biobrick part, CP29-RBS-aeBlue ([https://parts.igem.org/Part:BBa_K1033280 BBa_K1033280]), was used and assembled with the transporter genes.
-[[File:Mingdaophil1026-4.jpeg|600px|center]]
+[[File:Mingdaophil1026-4.jpeg|450px|center]]
+To measure glucose uptake by the engineered ''E. coli'' expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34ug/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.
+[[File:Mingdaophil1026-5.png|500px|center]]
+Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.
+[[File:Mingdaophil1026-6.png|600px|center]]
+Fig. 3 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was greater in Na+/Glu group than in control group with 1.2 times difference.
+[[File:Mingdaophil1026-7.png|700px|center]]
+In our results, glucose absorption by Na+/glucose transporter expressing bacteria was achieved to 97% after 4 hours with 1.2 times enhanced efficiency compared to the normal E. coli. To further increase the rate of glucose uptake, one may think about the glucose metabolism or conversion to other materials when entering into the cell.
@@ Line 33: / Line 46: @@
 === Reference ===
 . Glucose Galactose Malabsorption. American Journal of Physiology - Gastrointestinal and Liver Physiology 1998;275:G879-G882
 . Functional Properties and Genomics of Glucose Transporters. Curr Genomics. 2007;8(2): 113–128.
 . The SLC2 (GLUT) Family of Membrane Transporters. Mol Aspects Med. 2013;34(0): 121–138.
 . Glucose and Glycolysis Are Required for the Successful Infection of Macrophages and Mice by Salmonella enterica Serovar Typhimurium. Infect Immun. 2009;77(7): 3117–3126.