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

To evaluate the expression of celD-estA protein fusion, expression experiments by arabinose induction were carried on. CelD-estA construct was used to transformed the Escherichia coli strains BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783. The E. coli strains BL21SI, Rosetta Gami, XL1 Blue, and C43 were obtained from Invitrogen, Novagen, Agilent and Lucigen, respectively, and the strain BW27783 was donated by Tec-Monterrey 2010.

The E. coli strains containing the celD-estA construct and non-transformed strains as negative controls were cultured in 6 mL of LB Miller broth. The initial optical density at 600 nm (OD₆₀₀) was 0.1, from there the batch cultures were incubated at 37°C until an OD₆₀₀ of 0.6 was attained. The expression was induced with 0.1mM of L-arabinose and the temperature of postinduction was changed to 30 °C. Culture samples collected from the bioreactor were harvested by centrifugation. Half the volume was used for the whole cell assay and the other half was processed with Clontech x-Tractor kit (Clontech) to obtain the soluble and insoluble fractions of each strain. Both fractions were separated by a 10% SDS-PAGE and visualized with GelCode Blue Stain Reagent (Thermo).

All the insoluble fractions of transformed strains have a significant amount of a protein that matches the predicted weight of our chimeric construct (100kDa), in comparison to their negative controls (insoluble fraction of wild type lysates)(Figure 1). There is also no significant visual difference between each induced strain. According to Clontech’s buffer kit user manual, our protein could be trapped in the pellet (insoluble phase) because of its high molecular weight (100kD > 40kD) and because it is a membrane- bound protein that can form multiprotein complexes. And as we did not use Clontech’s TALON CellThru for direct purification from crude cell lysates (unclarified cell lysates), which is the solution proposed by the user manual in order to further solubilize proteins. Unclarified cell lysates were not carried on due to the objective of the experiment was know the expression of celD-estA construct. Future research includes identification of protein membrane display by periplasm extraction.

Figure 1. Protein profile of cell lysates from culture experiments of E. coli Bl21 SI, C43, XL1 Blue, Rosetta Gami and BW27783. (a) BL21 TNI (1)IF (2)SF. C43 TI (3)IF (4)SF - TNI (5)IF (6) SF - WT (7)IF (8)SF. (9)PMWM. Xl1 blue, TI (10)IF (11)SF - TNI (12)IF (13) SF - WT (14)IF (15)SF. (b) Rosetta Gami TI (1)IF (2)SF - TNI (3)IF (4) SF - WT (5)IF (6)SF. BW27783 TI (7)IF (8)SF - TNI (9)IF (10) SF - WT (11)IF (12)SF.(13) PMWM. BL21SI, TI (14)IF (15)SF. TI – transformed and induced. TNI – transformed and no induced, WT – wild type (C-). SF – soluble fraction. IF – insoluble fraction. PMWM – protein molecular weight marker

With this information we can only confirm the production of our protein. Further enzymatic assays were performed to know if the protein is in an active form.

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.