From 2007.igem.org

System Parts

educatETH E.coli consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our In the Lab page!). Four of them (4,5 and 8,9) form together two functional system units, but they have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids. Are you interested in the structure, mode of action or purpose of individual parts? Just click on the specific links in the third column. This will directly guide you to the entries in the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. All 11 educatETH E.coli parts are listed in the following table:

Single System Parts

1	TetR production	[http://partsregistry.org/Part:BBa_I739001 BBa_I739001]	constitutive subsystem
2	LacI production	[http://partsregistry.org/Part:BBa_I739002 BBa_I739002]	constitutive subsystem
3	LuxR production	[http://partsregistry.org/Part:BBa_I739003 BBa_I739003]	constitutive subsystem
4	1st half of P22 cII / EYFP production	[http://partsregistry.org/Part:BBa_I739004 BBa_I739004]	reporting subsystem
5	2nd half of P22 cII / EYFP production	[http://partsregistry.org/Part:BBa_I739005 BBa_I739005]	reporting subsystem
5new	2nd half of P22 cII / EYFP production	[http://partsregistry.org/Part:BBa_E0430 BBa_E0430]	reporting subsystem
6	cI production	[http://partsregistry.org/Part:BBa_I739006 BBa_I739006]	learning subsystem
7	P22 cII production	[http://partsregistry.org/Part:BBa_I739007 BBa_I739007]	learning subsystem
8	1st half of cI / ECFP production	[http://partsregistry.org/Part:BBa_I739008 BBa_I739008]	reporting subsystem
9	2nd half of cI / ECFP production	[http://partsregistry.org/Part:BBa_I739009 BBa_I739009]	reporting subsystem
10	RFP production	[http://partsregistry.org/Part:BBa_I739010 BBa_I739010]	reporting subsystem
11	GFP production	[http://partsregistry.org/Part:BBa_I739011 BBa_I739011]	reporting subsystem

Those 11 basic parts have been further assembled into intermediates and composites, which in turn enable the system to operate:

Composite System Parts

2+3	lacI + luxR production	[http://partsregistry.org/Part:BBa_I739012 BBa_I739012]	constitutive subsystem
1+2+3	tetR + lacI + luxR production	[http://partsregistry.org/Part:BBa_I739013 BBa_I739013]	constitutive subsystem
4+5	P22 cII + EYFP production	[http://partsregistry.org/Part:BBa_I739015 BBa_I739015]	reporting subsystem
8+9	cI + ECFP production	[http://partsregistry.org/Part:BBa_I739016 BBa_I739016]	reporting subsystem
(4+5)+(8+9)	(P22 cII + EYFP) + (cI + ECFP) production	[http://partsregistry.org/Part:BBa_I739017 BBa_I739017]	reporting subsystem
6+7	cI + P22 cII production	[http://partsregistry.org/Part:BBa_I739018 BBa_I739018]	learning subsystem
10+11	RFP + GFP production	[http://partsregistry.org/Part:BBa_I739019 BBa_I739019]	reporting subsystem
(6+7)+(10+11)	(cI + P22 cII) + (RFP + GFP) production	[http://partsregistry.org/Part:BBa_I739020 BBa_I739020]	learning/reporting subsystem

Many of the above mentioned parts contain double promoters. This promoter constructs contain two independent classes of operator sites and can therefore be regulated by two different types of molecules. Double promoters form the basis of the multi-inducible toggle switch and hence the memory of our learning system. This concept could also help future projects in developing devices and systems that need extended regulation. In the following, we introduce a selection of first generation double promoters:

Double Promoters

1pro	cI negative / tetR negative promoter	[http://partsregistry.org/Part:BBa_I739102 BBa_I739102]	reporting subsystem
2pro	lacI negative / P22 cII negative promoter	[http://partsregistry.org/Part:BBa_I739103 BBa_I739103]	reporting subsystem
3pro	luxR/HSL positive / P22 cII negative promoter	[http://partsregistry.org/Part:BBa_I739104 BBa_I739104]	learning subsystem
4pro	luxR/HSL positive / cI negative promoter	[http://partsregistry.org/Part:BBa_I739105 BBa_I739105]	learning subsystem
5pro	tetR negative / P22 cII negative promoter	[http://partsregistry.org/Part:BBa_I739106 BBa_I739106]	reporting subsystem
6pro	cI negative / lacI negative promoter	[http://partsregistry.org/Part:BBa_I739107 BBa_I739107]	reporting subsystem

In order to test if the concept of the proposed double promoters is working, simple proof of concept (PoC) parts have been constructed. The PoC promoter, which shows strong similarites to [http://partsregistry.org/Part:BBa_I739102 BBa_I739102], consists of two TetR operator sequences linked to a constitutive promoter. In contrast to the other double promoters , the PoC promoter is only single regulated. The PoC intermediate is part of the PoC composite the concept can be tested with, that is, EYFP production. The p22cII coding region is included to ensure the functionality in multicistronic constructs.

Proof of Concept

1poc	PoC promoter	[http://partsregistry.org/Part:BBa_I739101 BBa_I739101]	proof of concept, no part of the system
2poc	PoC intermediate	[http://partsregistry.org/Part:BBa_I739014 BBa_I739014]	proof of concept, no part of the system
3poc	PoC composite	[http://partsregistry.org/Part:BBa_I739021 BBa_I739021]	proof of concept, no part of the system

Although the parts have been synthesized by GENEART and also were shipped in their high-copy plasmids, we desided to change the cloning vectors. This strategy is based on the fact that fluorescent proteins are potentially harmful to the cells. The parts containing DNA sequences coding for these reporter proteins are therefore supposed to be cloned in low-copy number plasmids. In contrast, the constitutive subsystem parts can be cloned into a well characterized and working high-copy plasmid. Summarized, the following plasmids have been used:

Plasmids

1vec	pBR322BB1	[http://partsregistry.org/Part:BBa_I739201 BBa_I739201]	high-copy cloning vector, ApR constitutive subsystem
2vec	pBR322BB2	[http://partsregistry.org/Part:BBa_I739202 BBa_I739202]	high-copy cloning vector, TcR
3vec	pCK01BB1	[http://partsregistry.org/Part:BBa_I739203 BBa_I739203]	low-copy cloning vector, CmR reporting subsystem
4vec	pACYC177BB1	[http://partsregistry.org/Part:BBa_I739204 BBa_I739204]	low-copy cloning vector, KmR learning and reporting subsystem

Two E. coli strains have been used in this project. In the beginning, only the Top10 strain was worked with. But in a later stage of the project, using JM101 cells seemed to be more productive since they are growing faster than the Top10 cells.

Cell Strains

1str	Top10	[http://partsregistry.org/Part:BBa_V1009 BBa_V1009]	chemically competent E.coli from Invitrogen
2str	JM101	[http://partsregistry.org/Part:BBa_I739301 BBa_I739301]	original blue/white cloning strain