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 9494 (+) control M10210 M10211 M10212 M10213 M10214 M10215 M10216 M10217 M10218 M10219 M10221 M10224 M102250ul 12 20 20 19 18 18 16 15 17 16 16 14 19 13.25ul 552 438 400 306 470 368 452 308 374 393 290 474 461 27.50ul 781 972 856 859 866 682 900 993 1068 1035 1138 1544 1258 461.0ul 913 2242 2029 1991 2155 1987 1986 2151 2237 2193 2007 2265 2128 58

arab induced

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

A1 A2 A3 A4 9494 1363 A1 9494 136350073 39037 80477 35266 119554 51386 40860 58326 5044352273 38172 89294 38318 119557 51469 48840 59589 51059

Induced Uninduced

SAFIRE II; Serial number: 807003897; Firmware: V 2.10 12/2007 Safire2; XFLUOR4SAFIREII Version: V 4.62nDate: 8/7/09Time: 16:08

Measurement mode: FluorescenceExcitation wavelength: 480 nmEmission wavelength: 578 nmExcitation bandwidth: 20 nmEmission bandwidth: 20 nmGain (Manual): 50Number of reads: 20FlashMode: High sensitivityIntegration time: 40 µsLag time: 0 µsPlate definition file: COS96fw.pdfZ-Position (Manual): 11019 µmTarget Temperature: 37 °CCurrent Temperature: 28.5 °C

Rawdata (RFU) Temperature: 28.5 °C

V Bottom

A1 A2 A3 A4 9494 1363 A1 9494 1363109 33 372 31 1167 29 51 35 29128 31 505 24 960 28 55 36 28

Induced UninducedFlat Bottom

A1 A2 A3 A4 9494 1363 A1 9494 136343 9 206 11 655 9 17 12 1050 11 287 6 633 11 24 13 11

Induced Uninduced

Flat Bottom

0

100

200

300

400

500

600

700

A1 A2 A3 A4 9494 1363 A1 9494 1363

Induced Uninduced

1

2

V Bottom

0

200

400

600

800

1000

1200

1400

A1 A2 A3 A4 9494 1363 A1 9494 1363

Induced Uninduced

1

2

Z-position: 11019um

Assay again, with more cells

• After 1 wash• Results

consistent with previous experiment: A1 and A3 were brighter

• Triplicates, all induced

A1A2A31363

9494

A4

Image-J Intensity Density after one PBS washA1 A2 A3 A4 9494(+) 1363(-)

73393 63353 106684 70413 106798 6661572566 63868 106570 69284 106868 6672773021 62020 105990 69527 106811 64286

Flat-Bottom optimized z-positionSAFIRE II; Serial number: 807003897; Firmware: V 2.10 12/2007 Safire2; XFLUOR4SAFIREII Version: V 4.62nDate: 8/7/09Time: 19:03

Measurement mode: FluorescenceExcitation wavelength: 480 nmEmission wavelength: 578 nmExcitation bandwidth: 20 nmEmission bandwidth: 20 nmGain (Manual): 50Number of reads: 20FlashMode: High sensitivityIntegration time: 40 µsLag time: 0 µsPlate definition file: COS96fb.pdfZ-Position (Calc. from Well E1): 5720 µmTarget Temperature: 37 °CCurrent Temperature: 28.3 °C

Flat-BottomA1 A2 A3 A4 9494(+) 1363(-)

115 18 507 22 2518 17115 18 519 21 2125 14120 19 537 20 2205 13

Flat-Bottom OD NormalizedA1 A2 A3 A4 9494(+) 1363(-)1236.559 88.88889 1872.922 76.20367 8244.925 58.905061256.831 83.91608 1922.222 71.72131 7162.117 48.225971202.405 118.8243 2205.339 69.15629 7824.698 50.56398

Flat-bottom 5100 z-positionSAFIRE II; Serial number: 807003897; Firmware: V 2.10 12/2007 Safire2; XFLUOR4SAFIREII Version: V 4.62nDate: 8/7/09Time: 19:05

Measurement mode: FluorescenceExcitation wavelength: 480 nmEmission wavelength: 578 nmExcitation bandwidth: 20 nmEmission bandwidth: 20 nmGain (Manual): 50Number of reads: 20FlashMode: High sensitivityIntegration time: 40 µsLag time: 0 µsPlate definition file: COS96fb.pdfZ-Position (Manual): 5100 µmTarget Temperature: 37 °CCurrent Temperature: 28.3 °C

Flat-BottomA1 A2 A3 A4 9494(+) 1363(-)

104 18 448 19 2220 14103 17 458 19 1868 15110 18 478 20 1942 13

Flat-Bottom OD NormalizedA1 A2 A3 A4 9494(+) 1363(-)

1118.28 88.88889 1654.969 65.81226 7269.155 48.510051125.683 79.25408 1696.296 64.89071 6295.922 51.670691102.204 112.5704 1963.039 69.15629 6891.412 50.56398

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 AssaysCellulases

Assays should measure the ability of Bacteria to degradeinsoluble 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 AssaysCellulases

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 Assaygliadin 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: 16979252

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