Difference between revisions of "Part:BBa K2230027"

Latest revision as of 06:05, 26 October 2017

CP29-RBS-aeBlue-RBS-crr-RBS-ptsG-TT/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 Mingdao iGEM 2017]) for more information.

Cloning

Promoter CP29-RBS-aeBlue (BBa_K1033280) was assembled with RBS-crr-RBS-ptsG-TT/pSB1C3 (BBa_K2230026)

Function

An active high-affinity glucose transporter system. Promoter CP29 is a constitutive and strong promoter which works well in both E. coli and Lactobacillus spp. The blue protein encoded by aeBlue gene acts as indicator for the gene expression in the transgenic strain. This device can help E. coli and Lactobacillus spp. efficiently take up and retrieve glucose in the environment.

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.

As shown in Fig. 3, the growth of E. coli expressing the PTS reporter (i.e., crr and ptsG genes) was seriously retardant. The E. coli overexpressing glucose transporter may lose viability because of the toxic metabolites produced in glycolytic pathway. Not surprisingly, the PTS overexpression bacteria was almost unable to absorb glucose.

Our data is consistent with the previous study that overexpressing glucose transporter genes do really harm E. coli partly due to disturbing glycolytic pathways (J Gen Microbiol., 1992).

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.

5. Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli. J Gen Microbiol. 1992;138(10):2007-14.

Sequence and Features