Template:SBB-Protocols Assay4

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=111355 PMCID: PMC111355
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=193771 PMCID: PMC193771

This protocol doesn't quite follow a straight line--it's going in circles. It needs to be revised with details about volumes, plasticware, temperatures, and shaking conditions. Note that the arabinose stock is 1000x at 100mg/mL. The final concentration you want is 100ug/mL.

1. Transform all cultures with RFP-plasmid
2. Seed cultures in V-bottom plate
You'll want to do starter cultures, grow them to saturation to normalize, and then dilute them into the V-bottom plate in arabinose media 3. Treat cultures with Arabinose at 100mg/John Wang
4. Identify colonies that display prominent flocculation
How long will you grow them? Will they be shaken or just incubated? What temperatures? 5. Proceed with quantitative measurement for chosen colonies

6a. Negative Control: pBca9495CA-Bca1363, Positive Control: pBca9145-Bca9494
7a. Grow select colonies and controls to an OD of 0.5 at 600nm
8a. Add 100mg/John Wang Arabinose and shake for 3 hours at 37°C
9a. Transfer 200 uL to 96-well plate and take OD at 20 minute intervals
10a. Normalize cell concentration against RFP after addition of 100uL of 2MSodium Hydroxide

6b. Negative Control: pBca9495CA-Bca1363, Positive Control: pBca9145-Bca9494
7b. Add 100mg/John Wang Arabinose and shake for 3 hours at 37°C
8b. Grow select colonies and controls to an OD of 0.5 at 600nm
uL/ 9b. Transfer 200 uL to 96-well plate and take OD at 20 minute intervals
10b. Normalize cell concentration against RFP after addition of Sodium Hydroxyl

Protocol: Revamped

1a. Transform DH10B cells with pBca1600-Bca1144 (RFP-plasmid)
1b. Transform other DH10B cells with:
-- 1. the IILK test plasmids: pBca9495CA-(M10218-M10225)
-- 2. the AG4 negative controls: pBca9495CA-(M10210-M10217)
-- 3. the Pbad negative control: pBca9495CA-Bca1363
2a. Pick transformed pBca1600-Bca1144 (RFP-plasmid) colonies then transform with each IILK test plasmid and negative controls.
3. Pick 2 colonies/plasmid and grow to saturation in 3mL LB in shaker at 37°C (12 hours)
-- use 24 well block
4. Dilute them 10-fold (10uL saturated cell culture into 90uL LB) and seed 2 replicates of each clone into a V-bottom plate and 2 replicates of each clone into a plate that will go into the shaker
-- Add Arabinose (100ug/mL) to 1 replicate/clone on each plate
5. Put the V-bottom plate into the incubator and the other plate into the shaker
6. Grow to saturation (12 hours)
7. Look for floculation in the V-bottom plate
7. Take OD (600nm and 750nm?)
-- Blank the spectrophotometer with LB
-- Measure OD-600, 750 of shaken cells
-- Measure OD-600, 750 of non-shaken cells if necessary (ie-no meaningful results from shaken cell measurements)
-- If do this, need to find good way to resuspend cells. Perhaps shake vigorously for 10 sec?
8a. Normalize cell concentration against RFP after addition of 100uL of 2M Sodium Hydroxide

NOTES

David De Renzy 15:39, 22 April 2009 (EDT)