Team:Tec-Monterrey/projectresults

There are several methods to prove the successful transportation of CelD and SacC on the outer membrane of Escherichia 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.

CelD + estA protein fusion profiles
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).

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 wide, 100kDa band for the Induced BW27783 lanes in the insoluble fraction. This phenomenon happens on every induced strain. 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!

Roseta Results pdf

The Escherichia coli strain, Rosetta Gami, was choosen as a host for the chimeric protein because it has an improved folding system.
We used the IUPAC Filter Paper Assay, to determine the activity of the cellulase. This method is based in the reduction of the DNS, generating a proportional colorimetric concentration.
The assay was assest to the whole-cells, but also, we lysated the cells and separated them in two main fractions: soluble and insoluble. We were expecting more activity in the insoluble because our protein has a transmembranal domain.
The negative controls of the assay were non-transformed cells.
All samples were treated equally in the assay.

In the “ Whole-cell cellulase Activity” chart, is observed that there is a difference in the glucose concentration between the Negative Control cells (C-) and the CelD+ estA cells. A t-student was done (sigma = 0.05) . The result was the rejection of the null hypothesis (Ho), this meant that the difference was significant.

In the “Cellulase Activity Cell Lysates” is analysed two fractions : Soluble ans Insoluble. As it can be seen, in both cases, there is a difference between the celD+estA cells and their respective Negative Controls (C-) . Also, it is noticed that there is a higher difference in the Insoluble fraction that in the Soluble one. A t-student was done (sigma = 0.05). The result was the rejection of the null hypothesis (Ho), this meant that the difference was significant.

Conclusions
• The difference in the glucose concentration between the celD+esta and their Negative Control (C-) were significant. • The celD is ancher to the estA and it is active.
• The activity in the Negative Control (C-) is due to the background signal.


Future Work
• Standarize all the variables of the UIPAC Filter Paper Assay and do more measurement with the samples.
• Change the LB medium to a Basal medium like M9 medium.
• Try another E. coli strain like XL1 Blue, C43, Bl21 SI and others
• Assest others activity assays like Benedict Method and HPLC.

SacC Expression Results

The final genetic contrustion for ompA + sacC was accomplished without the translation terminator sequence (BBa_K633015). Approximately 3 kb of the linealized plasmid ompA + sacC was detected in all lanes and 1.25 kb of restriction fragment was visualized in the lane 6. (Figure 5)

Figure 5. 0.7% Agarose Gel. (Lane 1: Negative control (non-digested plasmid), 2: Linealized plasmid of ompA+sacC with EcoRI, 3: Linealized with XbaI, 4: 1kb DNA Ladder, 5: Linealized with SacI, 6: Digested with NheI, 7: Linealized with SpeI, 8: Linealized with PstI)

A visible protein band of the expected molecular wight (62.8 kDa) of the fusion protein (ompA+sacC) could not be confirmed by SDS-PAGE (Figure 6). However, as Lee et al. (2004) have proven, the fusion protein could hardly be detected by Coomassie blue staining as its expression was below the detection level of the method used, our result may be due to the same reason.

Figure 6. Tris-Glycine SDS-Polyacrylamide Gel of ompA + sacC. Gel A visualizes the 1D protein profiles of the soluble and insoluble fractions of XL1 Blue Wild Type (lane 2, 3), induced XL1 Blue + ompA + sacC plasmid (lane 4, 5) and non-induced XL1 Blue + ompA + sacC (lane 6, 7), the soluble and insoluble fractions of Rosetta Gami Wild Type (lane 8, 9), non-induced Rosetta Gami + ompA + sacC plasmid (lane 10, 11) and induced Rosetta Gami + ompA + sacC (lane 12, 13), and the soluble and insoluble fractions of induced BW27783 + ompA + sacC (lane 14, 15). Gel B shows the 1D protein profiles of the soluble and insoluble fractions of induced BW27783 + ompA + sacC (lane 1, 2), the soluble and insoluble fractions of BL21 SI wild type (lane 4, 5), non-induced BL21 SI + ompA + sacC (lane 6, 7), and induced BL21 SI + ompA + sacC (lane 8, 9), the soluble and insoluble fractions of C43 wild type (lane 10, 11), non-induced C43 + ompA + sacC (lane 12, 13), and induced C43 + ompA + sacC (lane 14, 15).

SacC is active in whole cell assays!

Whole cells of the E. coli strain (BL21SI) +sacC+ompA produced a fructose concentration of 350.71±60.97 uM which is 149.36 uM higher than the negative control cells (Figure 7), a T-test with 2 tails and alpha value of 0.05 was carried out, and the null hypothesis of "the population means are the same" was rejected, indicating that there is difference between the fructose concentration in the control strain and those of the sample strains. And although further investigation is required, the evidence we have is a strong indicator that the enzyme is active in the outer membrane of E. coli.

Figure 7. SacC Activity Assay Graph. Each bar indicates the fructose concentration generated by the sacC activity of non-transformed BL21 SI (left) and those of the BL21 SI + ompA + sacC plasmid.

Further research will be focused on SDS-PAGE with a more efficient staining/blotting technique, expression of sacC fusing it with estA protein fragments, and more sacC enzymatic assays.

• 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.