strep assay 7 26 09 cellulase parts plate 1 cel3a and cel5b l.
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Strep Assay 7-26-09 Cellulase parts Plate 1: cel3A and cel5B. OD normalized and averaged . Plate 1: cel3A and cel5B. cel5B. cel3A. uninduced. empty. Uninduced = cel3A with azo1653, oprF, cl02365, VtA11 (A1-A4). comparing arabinose induced with non-induced composite parts.

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plate 1 cel3a and cel5b
Plate 1: cel3A and cel5B

cel5B

cel3A

uninduced

empty

Uninduced = cel3A with azo1653, oprF, cl02365, VtA11 (A1-A4)

comparing arabinose induced with non induced composite parts
comparing arabinose induced with non-induced composite parts

Induced parts in general had 2X more fluorescence than uninduced parts

slide4

After 2nd wash

cel3A

cel3A, cel5B

cel5B

Uninduced (cel3A)

experimental conditions
Experimental conditions

Two plates of all the cellulase parts were ran:

  • Induced for ~6-7 hours for plates 1 and 2
    • 1:25 cell dilutions
    • Total volume 520ul (should be 500ul)
  • Done in duplicates
  • Did not have + and – controls… :/, but did have uninduced samples
  • ODs taken after washes as well as before washes
  • 2 washes, with UV pics taken after every wash
  • Fluorescence data taken with V bottom plate
plate 2 cellulases cel6a and cel9a7
Plate 2: cellulases cel6A and cel9A

Pcryo_1225 AtD

TshA

yuaQ AtD

cel6A

cel9A

cel9A

uninduced

cel6A

Empty

Azo1653 AtD

Uninduced = cel3A with eCPX, TshA, yua, AIDA (B1-B4)

experimental observations
Experimental observations
  • Noticeable differences (by factor ~2) between induced and uninduced for both plates 1 and 2- so a bit promising
    • Pcryo_1225 with cel9A highest
    • yuaQ with cel9A lowest
  • Plate 2 was incubated for longer than 30min (for 1hour), so perhaps reason for larger differences between induced and uninduced – nonspecific binding
  • When compared with previous strep assay, the normalized fluorescence was much higher (e.g. >1000) than the ones for this assay (~500) – could be because of high copy number versus low copy number
  • Reasons why diff from last time’s assay:
    • Poorly displayed, no controls, tag not well exposed
some conclusions next steps
Some conclusions/next steps

Will run the strep assay again 7-28-09 with two cellulase parts (cel5B and cel9A) and the needle scFv and gliadin scFv

Some changes:

  • + and – controls (EC100D cells)
  • + control (DH10B)
  • Triplicates
  • PBS in empty wells
  • Take ODs before assay and then after washes
  • Total volume is 500ul
  • 1:10cell dilutions
  • 3 washes instead of 2
  • Take Tecan measurements with gain of 50 and also higher (for greater sensitivity) to 55 ish
  • Take note of DH10B + control’s brightness
slide10

After 1st wash

cel9A

cel6A

cel9A, cel6A, uninduced

After 2nd wash

cel9A

cel6A

cel9A, cel6A, uninduced

comparing fluorescence with previous strep assay and control with atds
Comparing fluorescence with previous strep assay (+ and – control with AtDs)

Sort of can’t since some of the fluorescence measurements were not OD normalized and also v bottom vs flat bottom

growth issues with the cells
Growth issues with the cells

After 6 hours of incubation ODs were still low (~.1-.2)

the ODs from the scFvs from 7-27(12hrs)

And also the ODs from 7-26 (6hrs)