New and improved outgrowth assay
overnight inoculation of bacteria into LB for 12hours to saturation. Induce w/arabinose in new media for 12hours until saturation.
Recipe for Tris Buffer: 20mM Tris HCl, 150mM NaCl, pH 7.5.
espA - use the non-FITC conjugated batch in the lab fridge. The concentration of protein should be quantified via BCA assay
constructs: Needle scFv with various displayers (14 total)
negative controls: 1363 (1), no pass (14), gliadin scFv (2). (17 total)
Prepping Maxisorp Plates: *Note: Do everything in quintuplets!* *will need to use 2 plates for all constructs and controls. Put 3/4 of the construct in one plate with gliadin and 1363 controls, and 1/4 of the constructs with nopass controls*
1. incubate for 1.5hrs in 50ul of espA with Tris (5ul espA and 45ul Tris)<-- *will change once espA protein is quantified using BCA assay* 2. wash 2X with Tris in plate washer. Use program "3.14" and run it twice. 3. add 100ul of 1mg/ml BSA in Tris to block the plate. 4. incubate for 2hours at 37C no shaking. 5. aspirate BSA out using "asp1" program in plate washer. flick out any remaining liquid.
6. transfer 100ul of cells into a 96well plate bottom plate to take OD600 to get an idea of how uniform of cells we are putting in. 7. From those cells, add 5ul bacteria in 95ul of 1mg/ml BSA in TRIS. Make the BSA-TRIS first and pipet into maxisorp, and then squirt 5ul of cells into the maxisorp plate. 8. incubate for 1 hour at 37C no shaking 9. aspirate media and bacteria, and wash 3X using the "3.14" program 3 times, flicked out the remaining liquid<br> 10. being very sterile, add 100ul of LB with appropriate (AC) antibiotics 11. take an OD600 at this point (time =0) 12a. placed in 37C incubator, no shaking 13a. check and Tecan plate after every hour 14. plot results against negative controls alternatively, use the Tecan to take interval measurements. some concerns: Edge effects, so cannot use the outer rim of 96well plates. If cover is on, need to account for condensation. Is it sterile inside Tecan? will need 2plates for all neg controls and constructs. After adding 100ul media with AC antibiotics, 12b. set up Tecan at 37C, no shaking, intervals of 1hours, program Tecan to output data in a certain format easy for graphing <--ask John about that 13b. put plate into Tecan overnight