Small Molecule Induction Characterization

Progress

Cloning	Transfection	Dox	Cytometry	Data Analyis
Midis			8/20

Procedure

TRE	Doxycycline	0.1nM	1nM	10nM	100nM	1000nM
LacI	IPTG	0.1mM	0.5mM	1mM	5mM	10mM
Cumate	Cumate	0.5ug/ml	1ug/ml	5ug/ml	10ug/ml	50 ug/ml
Cumate	Cumate	5uM	10uM	50uM	100uM	500 uM
EGSH	Ponasterone A	0.1uM	0.5uM	1uM	5uM	10uM

Small molecule inducers will be added 24 hours after transfection and 24 hours before cytometry as per standard procedure.

Notes:

Glucose in the media may be a problem with the lac system (glucose is a weak anti-inducer of the lac repressor). A paper found a two fold decrease in induction in cells cultured in glucose containing media compared to galactose substituted media.

Cumate Molecular Weight: 194 g/mol

50 ug --> (50*10^-6)g/194g/mol = 0.257*10^-6mol

50ug/ml = 0.257*10^-6mol/ml = 0.257*10^-3 mol/L = 0.257mM

Plate Map

Plate 1 (Control Plate)

HEK293 Untransfected	hEF1a:mKate 500ng 1000nM Dox	hEF1a:mKate 500ng 10mM IPTG	hEF1a:mKate 500ng 500uM Cumate	hEF1a:mKate 500ng 10uM Ponasterone A
hEF1a:eBFP 500ng

Plate 2

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

0nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

0.1nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

1nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

10nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

100nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:rtTA 100ng

TRE:mKate 300ng

1000nM Doxycycline

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

0mM IPTG

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

.1mM IPTG

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

.5mM IPTG

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

1mM IPTG

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

5mM IPTG

hEF1a:eBFP 100ng

hEF1a:LacI 100ng

hEF1a-LacO:mKate 300ng

10mM IPTG

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

0uM Cumate

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

5uM Cumate

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

10uM Cumate

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

50uM Cumate

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

100uM Cumate

hEF1a:eBFP 100ng

hEF1a:CymR 100ng

hEF1a-CuO:mKate 300ng

500uM Cumate

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

0uM Ponasterone A

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

0.1uM Ponasterone A

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

0.5uM Ponasterone A

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

1uM Ponasterone A

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

5uM Ponasterone A

hEF1a:eBFP 100ng

hEF1a:VgEcR-2A-RXR 100ng

EGSH:mKate 300ng

10uM Ponasterone A

Background

To determine the optimal level of expression of BCR components and Syk-TEVp, we are placing them under the control of inducible/repressible promoters. This experiment aims to characterize the activity of the promoters we are going to use: TREt, hEF1a-LacO, hEF1a-CuO, and EGSH.

The pLac system may not be ideal to modulate expression given it's quick on/off response. We looked into other inducible expression systems:

EGSH promoter: http://parts.igem.org/Part:BBa_K415507

Cumate promoter:

http://parts.igem.org/Part:BBa_K875001

RheoSwitch:

http://66.155.211.155/nebecomm/products_Intl/productE3000.asp

RheoSwitch uses a Gal4 activator and the gene to be expressed is under control of a Gal4 response element. We can't use that because we are already using gal4 transcriptional activation to mediate receptor activation response.

Approach

We are going to investigate the expression of mKate driven by each of the promoters with varying concentrations of inducer (dox) and repressor (IPTG).