Team:Tec-Monterrey/projectresults/methods

1) From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 37C in 5-6ml LB+AMP (or other selection) in a 15ml snap cap tube on a rotator or shaker at 250 rpm.
2) Dilute until 0.1 OD600 in 6ml LB+AMP is achieved
3) Grow 3-4 hours at 37 C in 15ml snap cap tube in a rotator until 0.6 to 1.0 OD600. Is reached
4) Prepare 1ml LB+AMP+0.1mM arabinose in a 15ml conical and pre-warm to 37 C about 10min before use.
5) Add pre-warmed 1ml LB+AMP+0.1 arabinose to the cell culture
6) Incubate 18 - 24 hours at 30ºC at 250 rpm.

1) Harvest the bacterial cell culture by centrifugation at 1,000–3,000 x g for 15 min at 4°C. Remove the supernatant.
2) Add 20 ml of xTractor Buffer to 1 g of cell pellet. Mix gently. Pipet the mixture up and down to fully resuspend pellet
3) Add 40 μl of 1 unit/μl DNase I solution and 200 μl of 100X lysozyme solution. Add EDTA- free protease inhibitor. Mix gently, pipetting up and down several times.
4) Incubate with gentle shaking for 10 min at room temperature. (If desired, you may incubate the solution at 4°C.)
5) Centrifuge the crude lysate at 10,000–12,000 x g for 20 min at 4°C. Carefully transfer the supernatant to a clean tube without disturbing the pellet.

For pellet homogenization, sonication was carried on adding water at half of initial xTractor buffer volume of each batch culture. Pulses of 5-seconds at level 2 (Branson Sonifier 150) were performed until pellet was resuspended.

The celD+estA construction was generated by joining the biobricks of the araC-P_BAD 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 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-P_BAD 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 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 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).