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

Kimchi and Turkeys

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 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

Z-position: 11019um

Assay again, with more cells After 1 wash Results consistent with previous experiment: A1 and A3 were brighter Triplicates, all induced A1A2A A4

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

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 Cellulases 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.

Functional Assays Cellulases 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?

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 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:

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