Template:SBB-Protocols Assay4

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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

Contents

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-660, 750 of shaken cells
-- Measure OD-660, 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

Extra Info

Flocculating activity = (1− A/B) × 100 (%)

where A is the turbidity measured at 660 nm of a sample and B is that of a control.

NOTES

Jennifer Bophy 14:41, 20 April 2009 (EDT)

Started the Cell-cell adhesion assay. Transformed pBjb1600-Bca1144 (RFP) into DH10B cells. Plated on Spec plates. These cells will eventually be co-transformed with the IILK and AG4 plasmids to test cell density at the end of our experiments.
We are also doing a round of clones without the RFP plasmids, but we did not transform any DH10B cells with plasmids because we are hoping that other groups will have extra colonies.

Pick colonies (3 of each), grow to saturation, dilute, ect.

Jennifer Bophy 15:47, 21 April 2009 (EDT)

Picked colonies into 24 well plate with 3mL of LB. First two columns (A1-D1 and A2-D2) are pBca9495CA-(M10218-M10225), the next two columns are pBca9495CA-(M10210-M10217). A5 is pBca9495CA-Bca1363 and B5 is pBca1600-Bca1144 (RFP-plasmid).

Do dilutions. Re-transform RFP cells with corresponding plasmids.

Jennifer Bophy 14:24, 22 April 2009 (EDT)

Dilute cells into 96-well plate (to go into the shaker) and V-bottom plate (to go into the incubator). In the 96 well plate put in 1 mL of LB (with or without arabinose) and 100uL of saturated cell culture. In the V-bottom plate, put in 300uL of LB (with or without arabinose) and 30uL of saturated cell culture.
In V bottom plate:

A1-8=IILK with arabinose
B1-8=IILK without arabinose
C1-8=AG4 with arabinose
D1-8=AG4 without arabinose
E1=1363 with arabinose
E2=1363 without arabinose

In 96 well plate:

A1-8=IILK without arabinose
B1-8=IILK with arabinose
C1-8=AG4 without arabinose
D1-8=AG4 with arabinose
E1= 1363 without arabinose
E2= 1363 with arabinose

The RFP cells were also transformed with our plasmids of interest.

OD measurements, look at V-bottom plate, pick colonies.

David De Renzy 15:52, 22 April 2009 (EDT)

Transformation by heat shock on RFP DH10B:
8 IILK - man19-26
1 AG4 - man18
1 Pbad - Bca1363

Plated on half C-plates
-using 85μL of transformed cells

David De Renzy 12:00, 23 April 2009 (EDT)

Cell-Cell Adhesion Assay		OD Measurements			4/23/2009
							
w/ Arabinose & pipetting up/down 5 times							
Cell Type	Density(ug/mL)	OD-600	OD-750	Ratio	Notes		
IILK 1		52		1.104	0.732	1.51	Clumping		
IILK 2		54		1.147	0.78	1.47	Clumping		
IILK 3		61		1.282	0.883	1.45	No Clumping		
IILK 4		48		1.007	0.704	1.43	Clumps broke up easily		
IILK 5		54		1.129	0.751	1.5	No Clumping		
IILK 6		59		1.251	0.848	1.48	No obvious clumps		
IILK 7		57		1.2	0.85	1.41	No obvious clumps		
IILK 8		63		1.325	0.913	1.45	No Clumping		
AG4 2		68		1.435	1.015	1.41	Slight clumping		
AG4 3		64		1.36	0.936	1.45			
AG4 4		62		1.316	0.893	1.47			
AG4 5		58		1.231	0.831	1.48			
AG4 6		58		1.221	0.823	1.48			
AG4 7		60		1.256	0.848	1.48			
Pbad		81		1.709	1.238	1.38			
							
							
w/o Arabinose & pipetting up/down 5 times							
Cell Type	Density(ug/mL)	OD-600	OD-750	Ratio	Notes		
IILK 1		91		1.938	1.507	1.29			
IILK 2		91		1.926	1.512	1.27			
IILK 3		87		1.842	1.411	1.31			
IILK 4		90		1.898	1.456	1.3			
IILK 5		92		1.948	1.52	1.28			
IILK 6		86		1.828	1.387	1.32			
IILK 7		83		1.767	1.323	1.34			
IILK 8		85		1.795	1.346	1.33			
AG4 2		88		1.862	1.429	1.3			
AG4 3		87		1.851	1.405	1.32			
AG4 4		94		1.995	1.569	1.27			
AG4 5		88		1.869	1.422	1.31			
AG4 6		86		1.83	1.385	1.32			
AG4 7		86		1.823	1.369	1.33			
Pbad		89		1.882	1.434	1.31			
							
							
w/o Arabinose & No Mixing							
Cell Type	Density(ug/mL)	OD-600	OD-750	Ratio	Notes		
IILK 1		90		1.906	1.475	1.29			
IILK 2		89		1.89	1.481	1.28			
IILK 3		84		1.789	1.362	1.31			
IILK 4		88		1.865	1.426	1.31			
IILK 5		90		1.916	1.488	1.29			
IILK 6		85		1.791	1.359	1.32			
IILK 7		81		1.711	1.274	1.34			
IILK 8		83		1.765	1.318	1.34			
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