Modelado de procesos basado en reglas de negocio 1 Modelos relacionados a reglas de negocio

GALLEX is anin vivoreporter system used to measure the homo-interactions of a TM domain in

theE.coliinner membrane (26). The assay provides a semi-quantitative means of comparing the

relative strengths of homo-dimerisation for our TM domains with low complexity backgrounds in a biological membrane. DNA encoding of TM domains are genetically ligated between the gene for the DNA binding domain of theE. coliLexA transcription factor, and the gene for maltose binding protein, MBP. The MBP region resulting chimera is positioned in the periplasmic region and acts as a membrane anchor. Expression of the chimeric protein is controlled by the induction of the lac promoter with IPTG (Figure 3.1) and asE. coliis incapable of metabolising IPTG the concentration remains constant. on! lac! promoter/operator! MalE! LexA! IPTG induction!

Figure 3.1: GALLEX chimeras are expressed through the binding of the IPTG transcription factor to the lac promoter.

Transmembrane helix dimerisation is measured by binding of the dimeric LexA complex to the op+/op+promoter/operator region of theE. coligenome, resulting in repression of the reporter gene lacZ, and subsequent reduction in expression ofβ-galactosidase (Figure 3.2). This mechanism yields a system where the strength of the association between the TMs of interest is proportional to the inhibition ofβ-galactosidase.

MalE! MalE! MalE! MalE!

LexA! LexA! LexA! LexA! off!

op+/op+!

promoter/operator! Reporter gene _(lacZ)! !

Figure 3.2: The GALLEX chimera consisting of the dimer dependant DNA binding protein LexA, the TM domain, and the periplasmic anchor MBP. The TM domain interaction enables LexA dimerisation and subsequent repression ofβ galactosidase by binding to the op+promoter.

The strength of association between TM domains is visualised through a colourmetric assay. Solutions of lysed cells are inoculated with ortho-Nitrophenyl-β-galactosidase (ONPG), which hydrolyses into ortho-nitrophenol and galactose in the presence ofβ-galactosidase.

Controls were performed to ensure correct membrane insertion and orientation of the TM protein by transforming the ligated pBLM plasmid into competent NT326E. colicells. Some constructs were subjected to a sodium hydroxide wash to determine whether the protein was associating with the membrane, and all constructs were used in a MalE complementary assay to determine the correct orientation of the TM protein.

3.7.1 Chimera expression, insertion and orientation checks

The following sections explain the steps required to determine TM domain orientation, TM domain association with the membrane, and expression levels of chimeric protein.

3.7.1.1 SDS-PAGE

Quantification of the chimeric protein was determined using SDS-PAGE gels and western blotting. The 12% resolving layer of the polyacrylamide gels were prepared with 1.65 mL ddH2O, 2 mL 40%

acrylamide, 1.3 mL of 1.5 M Tris pH 8.8, 50µL 10% SDS, 50µL 10% APS, and 2µL TEMED. The 4% stacking layer was prepared from 1.4 mL ddH2, 0.266 mL 40% acrylamide, 0.26 mL 0.5 M

Tris pH 6.8, 20µ10% SDS, 20µ10% APS and 2µL TEMED. Samples were prepared by mixing cell solutions and lysed cell fractions with SDS sample loading buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.02% bromophenol blue, 5%β-mercaptonethanol) then boiled for at least 10 min. Running buffer was prepared with 25 mM Tris, 250 mM glycine, and 0.1% SDS. Gels were

run at 125 V, 36 mA, for 50 min.

3.7.1.2 Western Blotting

A stock of 1 L TBS (Tris-Buffered Saline) was prepared with 8 g NaCl, 0. g KCl and 3 g of Tris base. The pH was adjusted to 7.4 then 0.1% Tween20 was added to make TBST. Transfer of protein from gels to the nitrocellulose membranes was performed according to the instructions for the Invitrogen and Biorad kits. The resulting blots were blocked using 2% milk TBST solution for at least 1 hr on a rocker at room temperature. Nitrocellulose membranes were then washed with TBST for 3×5 min. The anti-MBP mouse primary anti-body was diluted to make a 1:4000 2% milk with TBST solution and applied to the blot. The blot was left to incubate at room temperature for 1 hr on a rocker. The primary anti-body was removed with a repeat of the 3×TBST washing. The final wash was removed and a 1:10000 dilution of an anti-mouse secondary anti-body in 2% milk and TBST solution applied to the blot and left to incubate at room temperature on a rocker. The wash was repeated as above. Protein bands were visualised by washing blots in 3 mL of BCIP/NBT reagent for 10 min. To stop the reaction the blot was washed in TBST for 10 min.

3.7.1.3 MalE complementary assay

A 5×M9 salts stock was prepared by dissolving 33.79 g Na2HPO4, 15 g of KH2PO4, 2.5 g NaCl

and 5.0 g NH4Cl in filter sterilised ddH2O to a final volume of 500 mL. A stock of 1 M MgSO4, 1

M CaCl2and 20 % w/v volume maltose were prepared in ddH2O and filter sterilised. 78 mL ddH2O

plates were prepared by adding 20 mL 5×M9 salts, 0.2 ml 1 M MgSO4, 2 mL 20 % maltose, and

0.01 mL 1 M CaCl2into the agar solution. Before pouring, the stock was inoculated with IPTG for

a final concentration of 0.01 M. An NT326 colony was picked from an LB ampicillin plate and streaked onto the maltose plate and then incubated for two to three days at 37◦C. A segment of the plate was left blank and another segment was streaked with untransformed NT326 cells as a negative control.

3.7.1.4 Sodium hydroxide extraction assay

5 ml of LB media was inoculated with 50µL cell culture and IPTG to a concentration of 0.01 M. The sample was incubated in a shaker until it reached an OD600 0.6 to 0.8. Cell cultures were

centrifuged at 3000 rpm for 10 min before being resuspended in 90µl ddH2O, 2.4µL MgCl2, 5 µL DNase (10 mg/ml) and 5µL lysozyme (10 mg/mL). Samples were left to equilibrate at room temperature for 1 hr then cooled on ice. 150µL ice cold ddH2O was added. A 125µL sample

was extracted for analysis (whole cell fraction) and set to one side on ice. The remaining cell solution was inoculated with 125µL ice cold 0.1 M NaOH and vortexed for 1 min. The sample was centrifuged at 14000 rpm for 15 min. Whole cell and supernatant fractions were mixed with 1 mL of 10 % TCA and left on ice for 30 min. Samples were centrifuged at 14000 rpm for 15 min and the supernatant was discarded. 1 mL of acetone was washed onto the surface of the pellet and left to incubate on ice for 5 min. A final centrifugation step at 14000 rpm for 10 min was performed. The supernatant was discarded and the pellet was allowed to air dry. The three fractions were resuspended in 80µL 1×protein sample buffer for quantification on a western blot.