ETHZ/Biology/Lab
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<center>[[ETHZ/Main_page | Main Page]] [[ETHZ/Biology | Biology Pespective]] [[ETHZ/Engineering | Engineering Perspective]] [[ETHZ/Meet_the_team | Meet the Team]] [[ETHZ/Internal | Team Notes]] [[ETHZ/Pictures | Pictures!]]</center><br> | <center>[[ETHZ/Main_page | Main Page]] [[ETHZ/Biology | Biology Pespective]] [[ETHZ/Engineering | Engineering Perspective]] [[ETHZ/Meet_the_team | Meet the Team]] [[ETHZ/Internal | Team Notes]] [[ETHZ/Pictures | Pictures!]]</center><br> | ||
- | <center><font size = '+ | + | <center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - In the lab ::. </b></font></center><br> |
In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section Problems might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Engineering | Engineering Perspective]]. | In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section Problems might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Engineering | Engineering Perspective]]. |
Revision as of 13:21, 15 October 2007
![Eth zh logo 2.png](/wiki/images/7/7e/Eth_zh_logo_2.png)
In this page, you can find information on laboratory conducted to construct EducatETH E.coli. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH E.coli at your lab, the section Problems might be useful to you. If you want to see the whole biological design of the system, please visit the Engineering Perspective.
Contents |
.:: List of system building blocks ::.
Here is a list of all the registry parts we used as bulding blocks for our system parts. This list has to be updated and extended for the new, concatenated parts that the ETH Zurich has submitted to the registry.
- [http://partsregistry.org/Part:BBa_B0034 B0034]
- [http://partsregistry.org/Part:BBa_R0062 R0062]
- [http://partsregistry.org/Part:BBa_R0053 R0053]
- [http://partsregistry.org/Part:BBa_J23100 J23100]
- [http://partsregistry.org/Part:BBa_J37033 J37033]
- [http://partsregistry.org/Part:BBa_E0434 E0434]
- [http://partsregistry.org/Part:BBa_B0015 B0015]
- [http://partsregistry.org/Part:BBa_Q04400 Q04400]
- [http://partsregistry.org/Part:BBa_R0010 R0010]
- [http://partsregistry.org/Part:BBa_E0422 E0422]
- [http://partsregistry.org/Part:BBa_R0040 R0040]
- [http://partsregistry.org/Part:BBa_R0051 R0051]
- [http://partsregistry.org/Part:BBa_Q04121 Q04121]
- [http://partsregistry.org/Part:BBa_C0053 C0053]
- [http://partsregistry.org/Part:BBa_Q04510 Q4510]
.:: Cloning plan::.
.:: Parts assignment into plasmids::.
Three plasmids are used for the EducatETH E.coli system parts as follows:
plasmid | resistance | copy type | contents | comments |
---|---|---|---|---|
pbr322 | ampicillin | high | 1,2,3 | constitutive subsystem |
pck01 | chloramphenicol | low | 4,5,8,9 | reporting subsystem |
pacyc177 | kanamycin | low | 6,7,10,11 | learning subsystem, reporting subsystem |
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.
.:: Linkers::.
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:
Linker | Plasmid |
---|---|
pbr322-1 | pbr322 |
pbr322-2 | pbr322 |
pbr322-3 | pbr322 |
pbr322-4 | pbr322 |
pck01 | pck01 |
pck01-2 | pck01 |
pacyc177-1 | pacyc177 |
pacyc177-2 | pacyc177 |
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.
.:: Procedure::.
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted before the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the donor and the plasmid accepting the new part will be referred to as the acceptor. Composite pars made of parts a and b are denoted a.b.
.::Plasmid 1 (pbr322ap)::.
- Put parts 1,2,3 in pbr322ap plasmids.
- Merge plasmid containing part 2 (donor) with plasmid containing part 3 (acceptor). You should get a plasmid containing a 2.3 composite part.
- Merge plasmid containing part 1 (donor) with plasmid containing composite part 2.3 (acceptor). You should get a plasmid containing a 1.2.3 composite part.
.::Plasmid 2 (pck01cm)::.
- Put parts 4,5,8,9 in pck01cm plasmids.
- Merge plasmid containing part 4 (donor) with plasmid containing part 5 (acceptor). You should get a plasmid containing a 4.5 composite part.
- Merge plasmid containing part 8 (donor) with plasmid containing part 9 (acceptor). You should get a plasmid containing a 8.9 composite part. Note: this step can be done simultaneously with the above.
- Merge plasmid containing composite part 4.5 (donor) with plasmid containing composite part 8.9 (acceptor). You should get a plasmid containing a 4.5.8.9 composite part.
.::Plasmid 3 (pacyc177km)::.
- Put parts 6,7,10,11 in pacyc177km plasmids.
- Merge plasmid containing part 6 (donor) with plasmid containing part 7 (acceptor). You should get a plasmid containing a 6.7 composite part.
- Merge plasmid containing part 10 (donor) with plasmid containing part 11 (acceptor). You should get a plasmid containing a 10.11 composite part. Note: this step can be done simultaneously with the above.
- Merge plasmid containing composite part 6.7 (donor) with plasmid containing composite part 10.11 (acceptor). You should get a plasmid containing a 6.7.10.11 composite part.
.:: Lab book ::.
.:: Week 1 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 06. Aug. 2007 |
| Sylke Raphael Stefan Markus Martin Christos Joe | |
Tue, 07. Aug. 2007 |
| Sylke Raphael Stefan Markus Martin Christos Joe | |
Wed, 08. Aug. 2007 |
| Raphael Stefan | |
Thu, 09. Aug. 2007 |
| Raphael Stefan Martin Christos Joe | |
Fri, 10. Aug. 2007 |
|
Christos | |
Sat, 11. Aug. 2007 | no labwork | ||
Sun, 12. Aug. 2007 | labwork cancelled |
.:: Week 2 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 13. Aug. 2007 start at 3 pm |
|
| Martin Markus Christos Tim |
Tue, 14. Aug. 2007 | Morning Shift:
Evening Shift:
| Morning Shift:
Evening Shift:
| Morning Shift (9am-1pm?): Markus Tim Evening Shift (5pm-...): Martin Christos |
Wed, 15. Aug. 2007 |
|
| From 12: Martin Markus |
Thu, 16. Aug. 2007 |
|
|
Markus |
Fri, 17. Aug. 2007 |
|
| Martin |
Sat, 18. Aug. 2007 | |||
Sun, 19. Aug. 2007 |
.:: Week 3 ::.
Little rearrangements of the parts. Planning of the sequences to order them.
.:: Week 4 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 27. Aug. 2007 | |||
Tue, 28. Aug. 2007 | |||
Wed, 29. Aug. 2007 | |||
Thu, 30. Aug. 2007 | |||
Fri, 31. Aug. 2007 | |||
Sat, 01. Sept. 2007 |
|
| Stefan |
Sun, 02. Sept. 2007 |
|
| Stefan |
.:: Week 5 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 03. Sept. 2007 |
|
| Martin Stefan |
Tue, 04. Sept. 2007 |
|
| Martin Christian |
Wed, 05. Sept. 2007 |
|
| Martin |
Thu, 06. Sept. 2007 |
|
| Christian |
Fri, 07. Sept. 2007 |
|
| Martin |
Sat, 08. Sept. 2007 | |||
Sun, 09. Sept. 2007 |
.:: Week 6 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 10. Sept. 2007 |
all digests o/n |
Christian | |
Tue, 11. Sept. 2007 |
|
|
Christian |
Wed, 12. Sept. 2007 |
|
Christian | |
Thu, 13. Sept. 2007 |
|
Christian | |
Fri, 14. Sept. 2007 |
|
*pBR322-MCS (Tet-selection) clone2 positive |
Christian |
Sat, 15. Sept. 2007 | |||
Sun, 16. Sept. 2007 |
.:: Week 7 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 17. Sept. 2007 |
|
|
Christian |
Tue, 18. Sept. 2007 |
|
> no DNA on pACYC177 digest-gel, only degradation smear
|
Christian |
Wed, 19. Sept. 2007 |
| ||
Thu, 20. Sept. 2007 | |||
Fri, 21. Sept. 2007 | |||
Sat, 22. Sept. 2007 | |||
Sun, 23. Sept. 2007 |
.:: Week 8 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 24. Sept. 2007 | |||
Tue, 25. Sept. 2007 | |||
Wed, 26. Sept. 2007 | |||
Thu, 27. Sept. 2007 | |||
Fri, 28. Sept. 2007 | |||
Sat, 29. Sept. 2007 | |||
Sun, 30. Sept. 2007 |
.:: Week 9 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 1. Oct. 2007 | |||
Tue, 2. Oct. 2007 | |||
Wed, 3. Oct. 2007 | |||
Thu, 4. Oct. 2007 | |||
Fri, 5. Oct. 2007 | |||
Sat, 6. Oct. 2007 | |||
Sun, 7. Oct. 2007 |
.:: Week 10 ::.
Date | TODO's | Completed | People |
---|---|---|---|
Mon, 8. Oct. 2007 | |||
Tue, 9. Oct. 2007 | |||
Wed, 10. Oct. 2007 | |||
Thu, 11. Oct. 2007 | |||
Fri, 12. Oct. 2007 | |||
Sat, 13. Oct. 2007 | |||
Sun, 14. Oct. 2007 |