Regulatory
HSP

Part:BBa_K338001

Designed by: 2010 Caltech iGEM Team   Group: iGEM10_Caltech   (2010-10-24)
Revision as of 07:25, 17 October 2018 by Adrireq (Talk | contribs)

Heat Shock Promoter (HSP)

This is a modified BBa_K112400 heat shock promoter in BBa standard.


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.

Contribution

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez, Carolina Ropero
Summary: We adapted the part to be able to assemble transcriptional units with the Golden Gate assembly method
Documentation:

In order to create our complete part collection of parts compatible with the Golden Gate assembly method, we made the part BBa_K2656001 which is this part adapted to the Golden Gate technology.

Sequence and Features BBa_K338001 SequenceAndFeatures

[edit]
Categories
//rnap/prokaryote/ecoli/sigma32
//direction/forward
//chassis/prokaryote/ecoli
//promoter
//regulation/positive
Parameters
negative_regulators
positive_regulators