Part:BBa_K2230022

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.

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.

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]