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Strep Assay 7-26-09 Cellulase parts Plate 1: cel3A and cel5B

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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Strep Assay 7-26-09 Cellulase parts Plate 1: cel3A and cel5B

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  1. Strep Assay 7-26-09 Cellulase partsPlate 1: cel3A and cel5B OD normalized and averaged

  2. Plate 1: cel3A and cel5B cel5B cel3A uninduced empty Uninduced = cel3A with azo1653, oprF, cl02365, VtA11 (A1-A4)

  3. comparing arabinose induced with non-induced composite parts Induced parts in general had 2X more fluorescence than uninduced parts

  4. After 2nd wash cel3A cel3A, cel5B cel5B Uninduced (cel3A)

  5. Plate 2: cellulases cel6A and cel9A

  6. 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

  7. 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)

  8. 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

  9. 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

  10. After 1st wash cel9A cel6A cel9A, cel6A, uninduced After 2nd wash cel9A cel6A cel9A, cel6A, uninduced

  11. 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

  12. 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)

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