Template:BioE140LSpr09-Autoaggregation

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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 

Replicates were grown up without arabinose to test for flocculating activity due to basal levels of protein expression.

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.
--Taniguchi et al

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 fromTaniguchi et al. 

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:
Chart.jpg

While pipetting the saturated cell culture from the 96-well blocks into the spectrophotometer cuvettes, we made the following observations:
WITH ARABINOSE: 

M10218   {<CPG_L6!}         saw one very large clump
M10219   {eCPX!}       
M10220   {upaG_short!}    many small clumps of cells that settled out of solution most in the 96-well block
M10221   {Ag43_short!}    very small faint clump
M10222   {espP(beta)!}      
M10223   {<ehaB!}             many small clumps
M10224   {CPompX!}          few clumps
M10225   {TshA!}                one small clump
*note: no visible clumps were seen for the "controls"

WITHOUT ARABINOSE: 

*no visible clumps were seen in the "samples" or "controls"

Analysis

The best carrier peptides for display of Leucine Zipper peptides are: {<CPG_L6!}, {<ipaG_short!}, and {<ehaB!}.

Basal levels of protein expression are not enough to see an autoaggregation phenotype.

In the future, the saturated cell cultures should also be mixed a few times before being pipetted into the cuvettes in order to ensure that all the cells (even those that have settled) are re-suspended and part of the OD measurements.

References

1. Kjærgaard, K., Schembri, M.A., Hasman, H., Klemm, P., Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens, J Bacteriol.(2000) 182: 4789–4796.
2. Taniguchi, M., Katoa, K., Matsuia, O., Pinga, X., Nakayamaa, H., Usukia, Y., Ichimuraa, A., Fujitaa, K., Tanakaa, T., Taruia, Y., Hirasawaa, E., Flocculating activity of cross-linked poly-γ-glutamic acid against bentonite and Escherichia coli suspension pretreated with FeCl3 and its interaction with Fe3+, J. of Biosci. & Bioeng. (2005) 100:207-211.
3. Veiga, E., Lorenzo, V., Fernández, L.A., Autotransporters as Scaffolds for Novel Bacterial Adhesins: Surface Properties of Escherichia coli Cells Displaying Jun/Fos Dimerization Domains, J Bacteriol. (2003) 185: 5585–5590.