Difference between revisions of "Template:BioE140LSpr09-Autoaggregation"

Revision as of 06:35, 11 May 2009

Autoaggregation: Cell-Cell Adhesion Assay

The purpose of this assay was to determine the ability of recombinant E.coli to aggregate when expressing IILK (leucine zipper) peptides on their outer membranes. The IILK peptide was displayed on the outer membrane via fusion to different displayer peptides. This assay tested the efficacy of the carrier proteins as fusions to functional IILK leucine zippers.
The following composite parts were tested during this assay:

M10218       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
M10219       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10221       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
M10222       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
M10224       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
M10225       {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}

Procedure

1. Transform DH10B cells with the following plasmids:
-- 1. the IILK test plasmids: pBca9495CA-(M10218-M10225)
-- 2. the AG4 negative controls: pBca9495CA-(M10210-M10217)
-- 3. the Pbad negative control: pBca9495CA-Bca1363
2. Pick 2 colonies per plasmid and grow to saturation in 3mL LB in shaker at 37°C (12 hours)
-- use 24 well block
3a. Dilute the saturated culture 10-fold (100uL saturated cell culture into 1mL LB)
-- Do two replicates of each clone into a 96-well block that will go into the shaker at 37°C
-- Add Arabinose (100ug/mL) to one replicate per clone
3b. Seed 2 replicates of each clone (10uL saturated cell culture into 100uL LB) into a V-bottom plate
-- Put plate in incubator at 37°C 6. Grow everything to saturation (12 hours)
7. Look for floculation in the V-bottom plate
7. Take OD measurements (600nm and 660nm) of cells grown in 96-well block
-- Blank the spectrophotometer with LB (or LB with arabinose)
-- Measure OD600 and 660 of shaken cells
-- When taking measurements, pipette 1mL out of 96-well blocks , then pipette each sample up and down 5 times in-order to ensure uniform mixing of each sample for an accurate OD measurements

Cell cultures with more autoaggregation will have less turbidity (lower OD measurements)

8. Flocculating activity = (1− A/B) × 100 (%)
-- where A is the turbidity measured of a sample and B is that of a control.

Results

V-bottom plates

Saw no clear evidence of flocculation in the V-bottom plates. We expected E.coli to remain suspended in the LB unless they autoaggregated, in which case they would clump at the bottom of the V-bottom plates because clumps of cells are heavier than individual cells. However, all of the E.coli settled at the bottom of the V-bottom plates (even the controls) making it impossible to distinguish a flocculating phenotype.

OD measurements

Flocculating Activity was calculated using the OD600 readings and the equation from ______. We defined A as the OD600 measurement of a "sample" (M10218-M10225). Samples were constructs with carrier proteins that had the IILK peptide fused to their N-termini. We defined B as the OD600 measurement of a control (M10210-M10217). Controls had the same carrier peptides as the samples ({CPG_L6!}, {<eCPX!}, ect.), but they displayed a different peptide ({<AG4>} instead of {<GS5-IILK>}.
Here is a chart of Flocculating activity with and without arabinose: