IGEM:IMPERIAL/2007/DNA Constructs

Construct Overview

Untitled

I	Part	Description	Status	Fate	Use in Constructs
	BBa_E0040	GFP	Not cloned	Ligate with T7 promoter	Test constructs prior to acGFP
	BBa_I13522	ptet-GFP	Not cloned	---	Basic construct for chassis characterization
	BBa_J5528	pBad-GFP	Not cloned	---	Test construct for pBad (CBD)
	BBa_J24677	ptet-luxR-plux-GFP	Not cloned	---	Basic construct for chassis characterization
	BBa_R0040	ptet	Not cloned	Ligate with acGFP, dsRFP, luxI	CBD final construct, BF AHL sender, Hrp test construct
	BBa_I0500	pBad	Not cloned	Ligate with acGFP, dsRFP	CBD final construct, Hrp test construct

Untitled 2

I	Part	Description	Status	Parents	Application
I	BBa_T9999	ptet-luxI	Dead	BBa_R0040 + BBa_F1610	AHL sender for BF

Parts from the registry

A	W	G	S	Name	Description	Plasmid	Resist	Copy	Size	Stock	Project
A	W			BBa_E0040	GFP	pSB1A2	AmpR	High	720bp	Well 5H, Plate1	Reporter
A	W			BBa_I13522	GFP under tetracyclin promoter	pSB1A2	AmpR	High	937bp	Well 15H, Plate2	Chassis characterization
A				BBa_J5528	GFP under arabinose promoter	pSB2K3	KanR	Low	2093bp	Well 6H, Plate3	CBD final construct
A	W			BBa_J24677	GFP under lux promoter, with luxR	pSB1A2	AmpR	High	1787bp	Well 18K, Plate3	Chassis characterization
A	W			BBa_R0040	tetracyclin promoter	pSB1A2	AmpR	High	54bp	Well 7O, Plate1	CBD constitutive promoter
A	W			BBa_I0500	arabinose promoter	pSB2K3	KanR	Low	1210bp	Well 9I, Plate2	CBD constitutive promoter
A	W			BBa_F2620	lux promoter, with luxR	pSB1A2	AmpR	High	1061bp	Well 6B, Plate1	BF final construct
A	W			BBa_F1610	luxI coding region	pSB1AK3	A+KR	High	798bp	Well 1B, Plate2	Use for BF sender
A	W			BBa_R0010	lac promoter	pSB1A2	AmpR	High	200bp	Well 7K, Plate1	Hrp constitutive promoter

Legend: A (Available), W (Working), G (Checked on gel), S (Sequenced)

Our Parts

A

W

G

S

Name

Description

Plasmid

Parents

Resist

Copy

Size

Stock

Project

Cell By Date

• Construct CBD 1
  • pT7 is currently in Biobricks Registry: BBa_J34814 (planning)
  • pT7 can be used with T7 RNAP in vitro and in veso, and it gives constitutive expression.

PT7 promoting GFP

• Construct CBD 2
  • Entire construct is currently in Biobricks Registry: BBa_I13522 (available), in pSB1A2 (AmpR)
  • pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.

Ptet promoting GFP

• Construct CBD 3
  • Entire construct is currently in Biobricks Registry: BBa_J5528 (available), in pSB2K3 (KanR)
  • Similar to part J5527 which has mRFP in place of GFP.
  • pBad can be used with E.coli RNAP in vitro, and it is inducible by arabinose.
  • To be used in veso, arabinose transport proteins have to be inserted in the phospholipid bilayer of the vesicles.

PBad promoting GFP

Biofilm Detector

• Construct BD 1
  • Entire part minus promoter is currently in Biobricks Registry: BBa_F1610 (available), in pSB1AK3 (AmpR, KanR)
  • pTet is currently in Biobricks Registry: BBa_R0040 (available), in pSB1A2 (AmpR)
  • pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.

Ptet promoting LuxI

• Construct BD 2
  • Entire part is currently in Biobricks Registry: BBa_J24677 (available), in pSB1A2 (AmpR)
  • Entire part minus GFP reporter system is currently in Biobricks Registry: BBa_F2620 (available), in pSB1A2 (AmpR)
  • GFP reporter system is currently in Biobricks Registry: BBa_I13504 (available)
  • pLux can be used with E.coli RNAP in vitro and maybe in veso, and it is repressed by LuxR but activated by AHL:LuxR complex.

Ptet promoting LuxR, Plux promoting GFP

• Construct BD 3
  • Entire part minus Hrp system is currently in Biobricks Registry: BBa_F2620 (available)
  • pHrpL can be used with E.coli RNAP in vitro and maybe in veso, and it is inducible by HrpR:HrpS:Sigma 54 complex.

Ptet promoting LuxR, Plux promoting HrpR and HrpS

PHrpL promoting GFP

Maybe, you meant LuxR instead of LuxI. Also, with the Hrp system, try to have a generic and modular approach when you define your constructs. PoPs_IN -> PoPs_OUT

Hrp Constructs

Promoter test constructs

Hrp PromA Promoter A is pLux BBa_R0062

Hrp PromB Two Test constructs will be made with different promoters, one with pBAD I0500 and one with pLac BBa_R0010

The assemblies displayed above will be used to test the properties of the promoters used in our devices. Both constructs use acGFP as their reporter.

Device 1 Construct

Hrp Dev1 Promoter A is BBa_R0062

A completed assembly of our first Hrp device that has only one input. We chose to add promoter (promoter A) and a fluorescence reporter (acGFP) as to make it functional and thus test it. A review of the actual device without the promoter\reporter can be found here .

Device 2 Constructs

Part 1	Hrp Dev2_1 Promoter B will be either BBa_I0500 or BBa_R0010	This includes promoter B which expresses HrpV and dsRFP at the same time.
Part 2	Hrp Dev2_2 Promoter A is BBa_R0062	This includes promoter A which expresses HrpR and HrpS eventually activating HrpL.Notice that this part is exactly the same as Hrp device 1 shown above.
The assemblies displayed above will be used to test the properties of the promoters used in our devices. Both constructs use fluorescence as their reporter.