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Basic Parts Update. Made 7 more parts since last week: cel9A, gliadin scFV, TevC, cel3A, OprF AtD, cl02365 AtD, Hia AtD Available passengers Gliadin scFv, Cub/Nub, Needle scFv, TevN/TevC, cellulases (cel3A, cel5B, cel9A, cel6A), SOD, Tir-M Available displayers

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basic parts update
Basic Parts Update
  • Made 7 more parts since last week: cel9A, gliadin scFV, TevC, cel3A, OprF AtD, cl02365 AtD, Hia AtD
  • Available passengers
    • Gliadin scFv, Cub/Nub, Needle scFv, TevN/TevC, cellulases (cel3A, cel5B, cel9A, cel6A), SOD, Tir-M
  • Available displayers
    • VirG AtD, yuaQ AtD, AIDA-1 AtD, (ehaB, eCPX, TshA, CPG_L6, CPG_L2, upaG_short, espP(beta)?), Hia AtD, Pcryo_1225 AtD, Hag AtD, cl02365 AtD, OprF AtD, azo 1653 AtD
  • In white box labeled “correct minipreps”
  • Put minipreps on stock plates
last two parts
Last two parts
  • Caspase(ig213) and mgfp-5(ig239)
  • mixing of forward and reverse oligos between the two
  • Purification gel looks good
  • Sequencing results:
    • Ig213 perfect! forward read, forgot to send in reverse read
    • Ig239 perfect!
strep assay
13 constructs from class: displayer, AG4/IILK, strep tag

96-well block – hard to see if all liquid aspirated out

Multichannel pipetting – inconsistent amount of liquid (and cells)

Strep Assay
strep assay1
Strep Assay
  • After 1 wash
  • A1= traA; A2=inv_short; A3=inv_long; A4=int-native
  • (+) control, A1, A3 had more brightness
  • Duplicates
  • C terminal displayers
  • Control is N terminal
  • Re-inoculated ~5hrs
assay again with more cells
Assay again, with more cells

After 1 wash

Results consistent with previous experiment: A1 and A3 were brighter

Triplicates, all induced

1363

A4

A3

A2

A1

9494

strep assay observations and next steps
Strep Assay - Observations and Next steps
  • Cell concentration was low after 5hours of incubation

– overnight incubation to saturation?

  • Used 1ul strep for all experiments
  • Flat bottom plate gave lower reading than V bottom plate, but the decrease was proportional
  • Uneven growth of cells, so need to normalize to OD
    • Control cells and the 13 constructs from 140L grew faster than the 4 C-term displayers
  • Tried – optimized Z-positions: 11019um, 5720um (optimized), 5100um
    • similar results
slide11
After OD-normalizing, A1 and A3 had similar fluorescence
  • Image J integration for the pictures

- the size and density of pellet seems to give a background so that smaller fluorescent pellets give similar readings as non-fluorescent larger pellets

  • Do 3 washes instead of 2 washes
  • Transfer from V to flat bottom necessary?
    • should stick to one plate type
functional assays

Functional Assays

Assays should measure the ability of Bacteria to degrade

insoluble cellulose.

Plate based assays exist:

-dyes form a colored complex with insoluble cellulose.

-Colonies that degrade insoluble cellulose remove color

from the plates.

-These methods are not very quantifiable.

Cellulases

functional assays1

Functional Assays

Assays have been preformed with purified enzyme product:

-cellulose in filter paper is degraded into reducing sugar.

-dinitrosalicylic acid is added to the solution

-absorbance at 600 nm is measured

These assays are preformed at ph 4.8 in an acidic buffer

-will the cellulases be active under non acidic conditions?

-will our cells survive under acidic conditions?

Cellulases

functional assay
Functional Assay

gliadin scFv: To test whether scFv binds to gliadin

  • ELISA
    • Coat plate with cells expressing surface scFv
    • Add 2% milk with TBS to block (to fill the bottom)
    • Add in gliadin that is tagged with fluorescent molecule (like FITC)
    • Wash 2X with TBS buffer
    • Use FACS to determine the amount of fluorescence
  • positive control
    • label cells with FITC and put through FACS
  • negative control
    • no plasmid in cell, so will not express scFv, so no binding to gliadin-FITC, therefore no fluorescence observed
    • or surface display a protein not specific for gliadin, so no fluorescence will result either
  • Can do similar binding assay with Tecan
functional assay1
Functional assay

Mgfp-5: to test whether mgfp adheres to surfaces

- Place solution of cells (3ul) expressing mgfp-5 on glass slide

- incubate at room temperature for 10minutes

- Do successive washes with PBS to wash cells out.

- look at cells on glass slide under light microscope

- measure cell density

- positive control: glass-adherent bacteria (E. coli B117)

- negative control: solution of cells not expressing mgfp-5

- PMID: 16979252

slide16
Modeling possibilities
    • Signal transduction system
    • Cell surface display (?)
    • Circuit required for tranducing signal (?)
    • Glue or cellulase (?)