BBa K338001

From 2010.igem.org


iGEM 2010



Home

People

Project Details

Protocols

Completed Systems

Notebook

Biosafety

Human Impact

References

Support


Caltech logo watermark.png

This is a modified K112400 heat shock promoter in BBa standard.


Contents

Usage and Biology

BioBrick Characterization

Effect of Heat Shock Temperature

DH5α cells containing HSP-GFP were incubated at 4°C, 25°C, 42°C, and 45°C separately for two hours. Measurements of the fluorescence level of GFP were taken using the plate reader at 20 minute intervals and were averaged over a 1 hour interval. The vertical axis is raw fluorescence units normalized by OD600. Normalized value of 4°C at t=0 was subtracted from all the values to show a relative difference.

Effect of Heat Shock Temperature.png


This experiment data suggests that heat shock promoter activity increases with heat shock temperature.

Effect of Heat Shock Duration

DH5α cells containing HSP-GFP were heat shocked at 42°C for 0 min (kept at room temperature), 5 min and 10 min separately. Measurements of the fluorescence level of GFP were taken using the plate reader at 15 minute intervals and were averaged over a 1 hour interval. The vertical axis is raw fluorescence units normalized by OD600.

Effect of Heat Shock Pulse Duration.png

The measurements suggest that a heat shock period of 10 minutes significantly increases the production of GFP, while a heat shock period of 5 minutes leads to a similar GFP production level as the untreated cells. Furthermore, the production of GFP shows a decreasing trend after 3 hours suggesting that the heat shock promoter was only activated for a certain time period. DH5α cells contained in the images below-visualized by an optical microscope with a GFP-specific filter- were incubated at 42°C for 10 minutes.

Images of Cells at t=0 min (Before Heat Shock)

T0 HSP-GFP 4(FITC).jpgT0 HSP-GFP 6(FITC).jpg

Control: cells containing only GFP at t=0

T0 GFP 12(FITC).jpg

Images of Cells at t=10 min

T10 HSP-GFP 27(FITC).jpgT10 HSP-GFP 29(FITC).jpg

Control: cells containing only GFP at t=10

T10 GFP 36(FITC).jpg

Images of Cells at t=60 min

T1hr HSP-GFP 39(FITC).jpgT1hr HSP-GFP 40(FITC).jpg

Control: cells containing only GFP at t=60

T1hr GFP 47(FITC).jpg

Glucose

An interesting observation was made: when adding 20 mM of glucose to the media at the time of growth,the cells are no longer morbidly elongated at the time of imaging.

Images of Cells at t=0 min (Before Heat Shock)

T0 HSP-GFP-glu 8(FITC).jpgT0 HSP-GFP-glu 10(FITC).jpg

Images of Cells at t=10 min

T10 HSP-GFP-glu 30(FITC).jpgT10 HSP-GFP-glu 32(FITC).jpg

Images of Cells at t=60 min

T1hr HSP-GFP-glu 44(FITC).jpg

NOTE: All images were captured at 100x magnification.

 
Caltech logo.png
Caltech footer.png


Locations of visitors to this page