Team:Mingdao/Basic Part
Basic Part
Part No. : BBa_K2230022
Type: Basic part
Cloning:
The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3. This part has been sequenced.
In order to express the genes in E. coli for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp1. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes.
Experiment & Result
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 34μg/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.
Result
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 result, glucose absorption efficiency was just slightly enhanced in Na+/glucose transporter expressing bacteria compared to general E. coli. To further increase glucose uptake, it is necessary to think about the glucose metabolism or conversion to other materials when entering into the cell.
Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point
© iGEM Mingdao 2017. Design: Kevin Li. All rights reserved.
