ETHZ/Biology/Lab

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Introduction Section Team Members Section Acknowledgments Section Site map Section

Introduction Section Model Overview Section Detailed Model Section Final Model Section Modeling Basics Page Mathematical Model Section FSM View Page Flip-Flop View Page Parameters Page

Introduction Section Test Cases Section Sensitivity Analysis Section

Introduction Section The Complete System Section System Phases Section System Parts Page Lab Notes Page

The ETH Zurich 07 Team Team Description

On this page, you may find information about how educatETH e.coli was implemented in the lab. More specifically, you will find information on the plasmid strains we used, the modifications we did to them in order to be compatible with the Biobrick library and our cloning plan. Moreover, in this page you may find here an (unfortunately not complete) electronic copy of our lab notebook. If you are here because you are interested in implementing educatETH E.coli in your lab, then our System Implementation and the System Parts pages may be of help to you!

Introduction

For all our cloning procedures we used standard protocols according to SAMBROOK and RUSSELL Molecular Cloning: A Laboratory Manual.

Strains

We used the following E. coli strains:

[http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29|E. coli Top10 (Invitrogen):]

You can find this strain at [http://partsregistry.org/Part:BBa_V1009 BBa_V1009]
This strain has a streptomycin resistance
Genotype: F’ {tetR}, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80 lacZ ΔM15, ΔlacX74, deoR, recA1, araD139 Δ(ara-leu)7679, galU, galK, λ-, rpsL,endA1, nupG
For further information please [http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29| click here]
References:
- Casdaban, M. and Cohen, S. (1980) J Mol Biol 138:179 PMID 6997493
- Grant, S.G.N. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 4645-4649 PMID 2162051

[http://openwetware.org/wiki/E._coli_genotypes#JM101|E. coli JM101:]

You can find this strain at [http://partsregistry.org/Part:BBa_I739301 BBa_I739301]
We call them Jimmys
This strain is the original blue/white cloning strain
Genotype: glnV44, thi-1, Δ(lac-proAB), F'[lacIqZΔM15 traD36 proAB+]
For further information please [http://openwetware.org/wiki/E._coli_genotypes#JM101| click here]
Reference:
- Messing, J. et al. (1981) Nucleic Acids Res. 9, 309; Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103

Plasmids

For our system we needed three plasmids with different origins of replication and antibiotic resistances. We decided to take low copy plasmids. We decided to use the following plasmids, which we wanted modify so that they would become compatible to the Biobrick Library multiple cloning site:

Basic plasmids

Plasmid	Resistances	Copy number	Origin	Map
pBR322	Ampicillin, Tetracyline	15-20 [1]	pMB1	pBR322 Map
pCK01	Chloramphenicol	5-12 [1]	pSC101	pCK01 Map
pACYC177	Ampicillin, Kanamycin	10-12 [1]	p15A	pACYC177 Map

Changes to the plasmids

In order to get the Biobrick multiple cloning site into the plasmids, we had to make several changes to the plasmids:

Plasmid	Changes	New name	New resistance	New Map
pBR322	Site directed mutagenesis: Changed the GCA codon of the PstI site of the bla gene into GTA Cloned in linker oligos (EcoRI/BamHI)	BBa_I739201	Ampicillin	BBa_I739201 Map
pBR322	Cloned in linker oligos (EcoRI/PstI)	BBa_I739203	Tetracycline	BBa_I739203 Map
pCK01	Site directed mutagenesis: Changed the ACT codon of the SpeI site in the origin of replication into ATT Cloned in linker oligos (AgeI/AseI)	BBa_I739202	Chloramphenicol	BBa_I739202 Map
pACYC177	Cloned in linker oligos (BamHI/PstI)	BBa_I739204	Kanamycin	BBa_I739204 Map

Cloning plan

Parts assignment into plasmids

The plan was to put the following parts into the three plasmids:

plasmid	resistance	copy type	contents	comments
[http://partsregistry.org/Part:BBa_I739201 BBa_I739201]	ampicillin	low	[http://partsregistry.org/Part:BBa_I739001 BBa_I739001(TetR) ], [http://partsregistry.org/Part:BBa_I739002 BBa_I739002(LacI) ], [http://partsregistry.org/Part:BBa_I739003 BBa_I739003(LuxR) ]	constitutive subsystem
[http://partsregistry.org/Part:BBa_I739202 BBa_I739202]	chloramphenicol	low	[http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII) ], [http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP) ], [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI) ], [http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP) ]	reporting subsystem
[http://partsregistry.org/Part:BBa_I739204 BBa_I739204]	kanamycin	low	[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI) ], [http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII) ], [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP) ], [http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP) ]	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.

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].

DNA assembly process ([1]) (Fig. 4)

Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted before the existing one. Composite parts made of parts a and b are denoted a.b.

Plasmid 1 (BBa_I739201)

Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] and BBa_I739201 plasmid with EcoRI/PstI and ligate them afterwards.
Digest [http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] and [http://partsregistry.org/Part:BBa_I739003 I739003(LuxR)] with XbaI/PstI
Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] in BBa_I739201 with SpeI/PstI.
Ligate digested [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] in BBa_I739201 with digested [http://partsregistry.org/Part:BBa_I739002 I739002(LacI)]. You get a plasmid containing a [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)].[http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] composite part.
Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)].[http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] with SpeI/PstI.
Ligate digested [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)].[http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] with digested [http://partsregistry.org/Part:BBa_I739003 I739003(LuxR)]. You get the completed [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)].[http://partsregistry.org/Part:BBa_I739002 I739002(LacI)].[http://partsregistry.org/Part:BBa_I739003 I739003(LuxR)] composite part in the BBa_I739201 plasmid.

Plasmid 2 (BBa_I739202)

Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)], [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] and the plasmid BBa_I739202 with EcoRI/PstI.
Digest [http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] and [http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] with XbaI/PstI.
Ligate digested [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] and digested plasmid BBa_I739202.
Ligate digested [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] and the plasmid BBa_I739202.
Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] in BBa_I739202 with SpeI/PstI.
Digest [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] in BBa_I739202 with SpeI/PstI.
Ligate digested [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] in BBa_I739202 with digested [http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)]. You get a plasmid containing a [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)].[http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] composite part.
Ligate digested [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] in BBa_I739202 with [http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)]. You get a plasmid containing a [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)].[http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] composite part.
Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)].[http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] with SpeI/PstI.
Digest [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)].[http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] with XbaI/PstI.
Ligate digested [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)].[http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] and digested [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)].[http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)]. You get the completed plasmid containing the [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)].[http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)].[http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)].[http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] composite part.

Plasmid 3 (BBa_I739204)

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.

References

[1] [http://www1.qiagen.com/faq/faqview.aspx?faqid=350&SearchText=&FaqCategoryId=0&MenuItemId=0&catalog=1&ProductLineId=1000228 QIAGEN FAQs]

[x] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]

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