< ETHZ | Biology
Revision as of 19:41, 24 October 2007 by Stefan Luzi (Talk | contribs)

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. All 11 educatETH E.coli are listed in the following table:

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

Composite System Parts
2+3 lacI + luxR production BBa_I739012 constitutive subsystem
1+2+3 tetR + lacI + luxR production BBa_I739013 constitutive subsystem
4+5 P22 cII + EYFP production BBa_I739015 reporting subsystem
8+9 cI + ECFP production BBa_I739016 reporting subsystem
(4+5)+(8+9) (P22 cII + EYFP) + (cI + ECFP) production BBa_I739017 reporting subsystem
6+7 cI + P22 cII production BBa_I739018 learning subsystem
10+11 RFP + GFP production BBa_I739019 reporting subsystem
(6+7)+(10+11) (cI + P22 cII) + (RFP + GFP) production 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 to the Registry of Standard Biological Parts:

Double Promoters
1pro cI negative / tetR negative promoter BBa_I739102 reporting subsystem
2pro lacI negative / P22 cII negative promoter BBa_I739103 reporting subsystem
3pro luxR/HSL positive / P22 cII negative promoter BBa_I739104 learning subsystem
4pro luxR/HSL positive / cI negative promoter BBa_I739105 learning subsystem
5pro tetR negative / P22 cII negative promoter BBa_I739106 reporting subsystem
6pro cI negative / lacI negative promoter 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 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 BBa_I739101 proof of concept, no part of the system
2poc PoC intermediate BBa_I739014 proof of concept, no part of the system
3poc PoC composite BBa_I739021 proof of concept, no part of the system

Although the parts have been synthesized by GENEART and were also 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 for 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 and should be cloned into a well characterized and working high-copy plasmid. Summarized, the following plasmids have been used:

1vec pBR322BB1 BBa_I739201 high-copy cloning vector, ApR
constitutive subsystem
2vec pBR322BB2 BBa_I739202 low-copy cloning vector, TcR
reporting subsystem
3vec pCK01BB1 BBa_I739203 low-copy cloning vector, CmR
reporting subsystem
4vec pACYC177BB1 BBa_I739204 low-copy cloning vector, KmR
learning and reporting subsystem
5vec pGA15 BBa_I739205 GENEART cloning vector, KmR

Cell Strains
1str Top10 BBa_V1009 chemically competent E.coli
from Invitrogen
2str JM101 BBa_I739301 original blue/white cloning strain