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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

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Basic Parts Update

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  1. 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

  2. Kimchi and Turkeys

  3. 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!

  4. 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

  5. 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

  6. Z-position: 11019um

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

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

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