"
Team:Tec-Monterrey/projectresults
From 2011.igem.org
There are several methods to prove the successful transportation of CelD and SacC on the outer membrane of E. coli. In this project, SDS-PAGE of entire cell culture samples, SDS-PAGE of membrane fraction samples, and measurement of enzyme activity of whole-cell-system without chemical or enzymatical purification operation have been considered in order to confirm the presence of active enzymes on the external membrane of E. coli.
As our second experiment, the protein profile for 6 expression strains (BL21 SI, BL21 STAR, XL1 Blue, C43, BW 27783 and Rosetta Gami) was produced at a lower temperatura (15°C) for 36h, which attenuated our bacterial metabolism and thus our transcription and traduction rate as well, securing the safe and secure folding of our proteins.
The order of wells on our gel is as follows:
In this gel we can see a tight, 100kDa band for the Induced BW27783 lanes, both on the soluble and insoluble fractions This phenomenon happens on every induced strain, and for Rosetta gami the soluble fraction contains clearly more protein than the insoluble fraction. This suggests that the 15°C induction produces better protein folding and fusion, due to the slowdown on E. coli 's relative to its speed at 30 or 37°C.
Click here to read our pdf file with results! The figure 1 shows that the whole-cell that was transformed with the device produced a concentration of glucose aproximately 2 mg per mL; this was confirmed with the negative control due to the lower absorbance data.
The figure 2 shows that the data of the velocity rate of the transformed cells could be related between them, meanwhile the data of the negative control showed no relation .
The insoluble and the soluble fraction of the ''''Rosetta Gami'''' were analized in the figures 3 to 6. In the insoluble fraction there was a bigger reaction rate than the soluble one. This is because the protein has a lot of hydrophobic regions.
The colorimetric method ( FPU, IUPAC) used can be improved, with a better temperature control and a better way of performing sample measurement; also there was not enough time for more conclusive results. But with these results we can assume that there IS enzyme activity.
Construction of genetic frame of OmpA fragment with SacC was confirmed by several digestion reactions and agarose gel electrophoresis.
Transformation of this construct was carried out into 5 differents expression strains of E. coli (BL21SI, XL1Blue, C43, Rosetta Gami, and BW27783) by chemical transformation with CaCl2. A isolated colony of each transformed strain was grown in LB until OD600 0.600, and the culture was induced with final concentration 0.01 mM of arabinose solution during 30 - 36 hours at 15°C.
Samples were processed with Clontech x-Tractor kit to obtain soluble and insoluble fraction of each strain.
Expected MW of fusion protein (OmpA + SacC) is 62.8 kDa, but successful expression of the construct could not be confirmed by SDS-PAGE method. However, as Lee et al. (2004) have proved that the fusion protein could hardly be detected by Coomassie blue staining because its expression level used to be very low, our result may be due to this reason. Further research should be focused on SDS-PAGE with more efficient staining/blotting technique and SacC enzyme assay.
Quantification of fructose was carried out with EnzyChrom Fructose Assay Kit from Medibena BioAssay Systems, which was donated by PhD Fernández. Transformed cell cultures (BL21SI, XL1Blue, C41, C43, Rosetta Gami, and BW27783) are induced with arabinose solution when their OD600 arrive at 0.600. After 30 hours of incubation at 15 °C, the cultures are centrifugated 5 min at 14,000 rpm. Supernatants are diluted with water and 20 uL of each sample is transfered into separate wells of 96-well plate. 56 µL Assay Buffer, 1 µL Enzyme, 14 µL PMS solution and 14 µL MTT solution are mixed and added to each well. After 1 hour of incubation at room temperature the plate is readed at 565 nm. The reaction is specific with fructose, so glucose and other sugar do not interfere. The color intensity is directly proportional to the fructose concentration.
• Lee SH, Choi JI, Park SJ, Lee SY & Park BC (2004) Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis. Applied and Environmental Microbiology. Vol.70(9):5074–5080.
In order to prove the presence of our proteic fusion (celD+estA) we ran several polyacrylamide gels to determine protein profiles on 6 different expression strains (BL21 SI, BL21 STAR, XL1 Blue, C43, BW 27783 and Rosetta Gami), to determine the correct variable combination, which would represent the best yield for our target protein. Said variables were time and induction temperature.
For our first assay, proteins were inducted in the BL21 SI, XL1 Blue, C43, BW 27783 and Rosetta Gami strains at 25°C for 12h. Once the induction time ended, cells were then lysed using the xTractor extraction kit, from Clontech, in order to obtain soluble and insoluble fractions.
The order of our polyacrylamide gels is as follows:
As it is seen, lanes corresponding to the insoluble fraction on transformed and induced strains show a thick band at around 100kDa* according to our molecular weight marker (Bio-Rad). Said band is not found in lanes corresponding to negative controls (wild-types and non-induced transformed cells).
Based on our results, we can assure that our protein was translated just as planned. Nevertheless, there’s a chance of finding a fraction of our protein as a part of an inclusion body. Then, we ran activity essays to test the correct folding of our protein.
*Our protein’s molecular weight was calculated by means of a predictive program based on the amino-acid sequence codified for our protein (http://www.scripps.edu/~cdputnam/protcalc.html).
