"
Team:Tec-Monterrey/projectresults/methods
From 2011.igem.org
The celD+estA construction was generated by joining the biobricks of the araC-PBAD promoter (BBa_I13458 and BBa_K206000), RBS+phoA signal peptide+celD (BBa_K633002) and linker+estA (BBa_K633001) with the biobrick standard assembly protocol ( Manual). The expected DNA fragment of the celD+estA construct was confirmed by several restriction endonuclease reactions, and used to transform 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).
The IUPAC Filter Paper Assay was used to determine the celD+estA activity. The E. coli strain , Rosetta Gami, was used as a host for the expression of the chimeric protein because it has an improved protein folding system. The assay was applied to the whole-cells, but these were also lysated with x-Tractor Cell lysis Buffer (Clontech), which separated them into soluble and insoluble fractions. The negative controls (C-) of all the samples were non-transformed cells. In the whole-cell cellulase activity experiment and in the cellulase activity of cell-lysates experiment, a t-test was done with an alpha of 0.05 to prove the hypothesis.
The SacC gene from Zymomonas mobilis was PCR amplified with the primers S1PSF: 5’-GAATT CGCGG CCGCT TCTAG AGGAG CTCAT GTTTA ATTTT AATGC CAGTC GC-3’, S1PSR 5’-CTGCA GCGGC CGCTA CTAGT AGCTA GCGTA TTTGC GACGA TCAGG G-3’. The amplification mixture for 50 mL contained 1U of Platinum Taq HF polymerase (Invitrogen), 60 mM Tris-SO4, 18 mM Ammonium Sulfate, 0.2 mM for each dNTP, 2 mM MgSO4, 2 mM of forward and reverse primers. PCR was performed in an MultiGene (Labnet) thermocycler using the following program: 94 ºC for 5 min, 35 cycles of 94 ºC for 45 s, 56.4 ºC for 30 s, and 68 ºC for 1 min, and finally an extension step at 68 ºC for 5 min. The PCR product was first sub-cloned in pGEM T Easy Vector (Promega) and added to the registry (BBa_K633003).
The ompA+sacC construction was generated by joining the biobricks of the araC-PBAD promoter (BBa_I13458 and BBa_K206000), RBS (BBa_B0034), lpp+ompA (BBa_K103006), and sacC (BBa_K633003) with the biobrick standard assembly protocol ( Manual). The expected DNA fragment of the ompA + sacC construct was confirmed by several restriction endonuclease reactions, and used to transform the E. coli strains BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783.
The E. coli strains containing the ompA+sacC construct and non-transformed strains as negative controls were cultured in 6 mL of media M9 with glycerol as its unique carbon source. 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 15 °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).
To determine the ompA+sacC activity, EnzyChromTM Fructose Assay Kit (kindly donated by PhD. Fernández) was used. The E. coli BL21 SI was used for the expression of the fusion protein. The assay was applied to the whole-cells, and non-transformed cells were used as a negative control. A t-test was done with an alpha value of 0.05 to compare fructose concentrations of each sample.
