Controls

Controls were employed to check the correct insertion of the chimeric protein constructs into theE. coli membrane as well as to ascertain the expression levels of the proteins.

2.9.4.1 Spheroplast Assay

An outgrowth was taken from an overnight culture of E. coli NT326 transformed with the GALLEX plasmid to be tested and grown in the presence of 0.01 mM IPTG to an OD600 of ~0.6. The cells from a 1.5 ml volume of this

outgrowth were harvested by centrifugation at 13,400 × g. for 2 minutes and resuspended in 0.5 ml buffer (100 mM tris-acetate pH 8.2, 0.5 M sucrose, 5 mM EDTA). 6 µl of 10 mg/ml lysozyme was added and incubated at 4 °C for 1 minute before adding 0.5 ml 4 °C dH2O and incubated for a further 4 minutes. 20 µl of 1 M

MgSO4was added before the resulting spheroplasts were pelleted at 13,400 × g and 4

°C for 2 minutes.

The spheroplasts were resuspended in 300 µl of buffer (10 mM HEPES pH7.6, 2 mM EDTA) and split into three equal sample fractions in 1.5 ml Eppendorf tubes. One fraction of the three, the whole spheroplast fraction, is then precipitated using trichloroacetic acid (TCA). 1 ml of 10% TCA is added and the sample incubated at 4 °C for 30 minutes before being pelleted at 13,400 × g for 15 minutes. All the supernatant is removed and 1 ml of acetone is added and incubated at 4 °C for 5 minutes before being pelleted again at 13,400 × g for 10 minutes. All the supernatant is removed and the pellet air dried.

One of the other fractions is treated with 2.68 µl of 19.6 mg/ml proteinase K and incubated at 4 °C for 30 minutes before being precipitated with TCA as described for the whole spheroplast fraction. The final fraction is freeze-thawed 5 times using a dry ice-ethanol bath and 37 °C water bath before added 2.68 µl of 19.6 mg/ml proteinase K and incubated at 4 °C for 30 minutes. This was then precipitated using TCA as described above.

All three fractions are then run on a standard 12% SDS-PAGE gel as described and the bands visualised by Western blotting using anti-MBP antibodies. MBP should be detected in all three fractions but due to the addition of proteinase K, which cleaves the MBP domain from the rest of the protein, in the last two, the band in this case will be seen at a lower molecular weight corresponding to just the MBP domain. This assay shows correct insertion because if the MBP domain is not exposed to the periplasm, proteinase K will not be able to cleave the domain in the second fraction in which whole spheroplasts are treated with proteinase K.

2.9.4.2 MalE Complementation Assay

The second control of insertion is growing E. coli NT326 cells, which lack native MBP, with the plasmids coding for the chimeric proteins on maltose minimal medium with no other carbon source. If the MBP domain is exposed to the periplasm, it should confer the ability to utilise the maltose as a carbon source on the cells and allow them to grow.

Maltose minimal medium is prepared by making sterilised 1 M MgSO4, 1 M

CaCl2 and 20% maltose (weight/volume) solutions. Agar and dH2O (final agar

concentration 15 g/l) was autoclaved and to this, 20 ml of 5X M9 salts, 0.2 ml 1 M MgSO4, 2 ml 20% maltose and 10 µl 1 M CaCl2was added. 5X M9 salts were made

according to the recipe in Molecular Cloning vol. 3 (64 g Na2HPO4·7H2O, 15 g

2.9.4.3 Surface Expression Dot Blot

An outgrowth was taken from an overnight culture ofE. coliand grown in the presence of 0.01 mM IPTG to an OD600of ~0.6. The cells from a 2 ml volume of this

outgrowth were harvested by centrifugation at 3,000 × g. for 2 minutes and resuspended in 0.5 ml buffer (100 mM tris-acetate pH 8.2, 0.5 M sucrose, 5 mM EDTA). 6 µl of 10 mg/ml lysozyme was added and incubated at 4 °C for 1 hour before adding 0.5 ml 4 °C dH2O and incubating for a further 4 minutes. 20 µl of 1 M

MgSO4was added before the resulting spheroplasts were pelleted at 3,000 × g. and 4

°C for 2 minutes. A 2 µl aliquot of this sample was pipetted directly onto a nitrocellulose membrane and allowed to dry. This membrane was then probed for MBP expression in the same way as the standard Western blot outlined in Section 2.7.2. Untransformed SU101 and NT326 cells were also included as controls to check for cell lysis.

2.9.4.4 NaOH Extraction

An overnight culture was inoculated 1:100 into fresh LB media with the

required antibiotics and 10 μM IPTG. The culture was grown until an OD600 of 0.6

was reached and then the cells were collected by centrifugation at 13000 × g for 1

minute. The cell pellet was resuspended in 90 μl H2O, 2.5 μl 1 M MgCl2, 5 μl DNase

(10 mg/ml), and 5 μl lysozyme (10 mg/ml). This mixture was incubated at room temperature for 1 hour and then cooled on ice. 150 μl of ice cold (~4 °C) H2O was

added and then 125 μl of this was taken as the whole cell fraction for analysis. 125 μl

ice cold (~4 °C) 0.1 M NaOH was added to the remainder and vortexed for 1 minute before centrifugation at 13000 × g for 15 minutes. The supernatant was taken as the

soluble protein fraction, and the pellet as the membrane protein fraction. The whole cell and the soluble protein fractions were precipitated using TCA as described above inSection 2.8.4.1.

2.9.4.4. Expression Check of Chimeric GALLEX Proteins

When performing the homo GALLEX assay a sample of cell culture was taken prior to running the assay. These samples were used to normalise the assay’s results to the protein expression level as well as to the cell density of the culture. The samples were normalised to OD600for cell density and then analysed by SDS-PAGE

and Western blotting using anti-MBP antibodies to specifically detect the chimeric GALLEX proteins as described inSection 2.8.2.