ETHZ/Biology/parts
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<center>[[ETHZ | Main Page]] [[ETHZ/Model | System Modeling]] [[ETHZ/Simulation | Simulations]] [[ETHZ/Biology | System Implementation]] [[ETHZ/Biology/Lab| Lab Notes]] [[ETHZ/Meet_the_team | Meet the Team]] [[ETHZ/Internal | Team Notes]] [[ETHZ/Pictures | Pictures!]]</center><br> | <center>[[ETHZ | Main Page]] [[ETHZ/Model | System Modeling]] [[ETHZ/Simulation | Simulations]] [[ETHZ/Biology | System Implementation]] [[ETHZ/Biology/Lab| Lab Notes]] [[ETHZ/Meet_the_team | Meet the Team]] [[ETHZ/Internal | Team Notes]] [[ETHZ/Pictures | Pictures!]]</center><br> | ||
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= System Parts = | = System Parts = | ||
- | + | EducatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab 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 <i>E.coli</i> parts are listed in the following table: | |
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | {| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | ||
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- | 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. | + | 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. |
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | {| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | ||
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- | Although the parts have been synthesized by GENEART and also were shipped in their high-copy plasmids, we | + | Although the parts have been synthesized by GENEART and also were shipped in their high-copy plasmids, we decided 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. Summarized, the following plasmids have been constructed: |
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | {| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | ||
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|- | |- | ||
! 2<sup>vec</sup> | ! 2<sup>vec</sup> | ||
- | | | + | | pCK01BB1 |
| [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] | | [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] | ||
- | | low-copy cloning vector, | + | | low-copy cloning vector, CmR<br>reporting subsystem |
| <partinfo>BBa_I739202 SpecifiedComponents</partinfo> | | <partinfo>BBa_I739202 SpecifiedComponents</partinfo> | ||
|- | |- | ||
! 3<sup>vec</sup> | ! 3<sup>vec</sup> | ||
- | | | + | | pBR322BB2 |
| [http://partsregistry.org/Part:BBa_I739203 BBa_I739203] | | [http://partsregistry.org/Part:BBa_I739203 BBa_I739203] | ||
- | | low-copy cloning vector, | + | | low-copy cloning vector, TcR |
| <partinfo>BBa_I739203 SpecifiedComponents</partinfo> | | <partinfo>BBa_I739203 SpecifiedComponents</partinfo> | ||
|- | |- | ||
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- | 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 | + | 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 grow faster than the Top10 cells. |
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" | {| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" |
Latest revision as of 19:56, 26 October 2007
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 Registry of Standard Biological Parts. All 11 educatETH E.coli parts are listed in the following table:
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 |
Those 11 basic parts have been further assembled into intermediates and composites, which in turn enable the system to operate:
2+3 | lacI + luxR production | BBa_I739012 | constitutive subsystem | |
---|---|---|---|---|
1+2+3 | tetR + lacI + luxR production | BBa_I739013 | constitutive subsystem | |
4+5new | P22 cII + EYFP production | BBa_I739015 | reporting subsystem | |
8+9 | cI + ECFP production | BBa_I739016 | reporting subsystem | |
(4+5new)+(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:
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.
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 also were shipped in their high-copy plasmids, we decided 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. Summarized, the following plasmids have been constructed:
1vec | pBR322BB1 | BBa_I739201 | low-copy cloning vector, ApR constitutive subsystem | |
---|---|---|---|---|
2vec | pCK01BB1 | BBa_I739202 | low-copy cloning vector, CmR reporting subsystem | |
3vec | pBR322BB2 | BBa_I739203 | low-copy cloning vector, TcR | |
4vec | pACYC177BB1 | 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 grow faster than the Top10 cells.
1str | Top10 | BBa_V1009 | chemically competent E.coli from Invitrogen | |
---|---|---|---|---|
2str | JM101 | BBa_I739301 | original blue/white cloning strain |