ETHZ/Biology/Lab
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# 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. | # 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. | ||
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# Put parts 6,7,10,11 in pacyc177km plasmids. | # Put parts 6,7,10,11 in pacyc177km plasmids. |
Revision as of 22:33, 24 October 2007
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:
- You can find this strain at 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 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
- Casdaban, M. and Cohen, S. (1980) J Mol Biol 138:179 PMID 6997493
- You can find this strain at 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 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 | |
pCK01 | Chloramphenicol | 5-12 [1] | pSC101 | |
pACYC177 | Ampicillin, Kanamycin | 10-12 [1] | p15A |
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 |
|
BBa_I739201 |
Ampicillin | |
pBR322 |
|
BBa_I739203 |
Tetracycline | |
pCK01 |
|
BBa_I739202 |
Chloramphenicol | |
pACYC177 |
|
BBa_I739204 |
Kanamycin |
Cloning plan
Parts assignment into plasmids
The plan was to put the following parts into the three plasmids:
plasmid | resistance | copy type | contents | comments |
---|---|---|---|---|
BBa_I739201 | ampicillin | low | BBa_I739001(TetR) , BBa_I739002(LacI) , BBa_I739003(LuxR) | constitutive subsystem |
BBa_I739202 | chloramphenicol | low | BBa_I739004(P22 cII) , BBa_E0430(EYFP) , BBa_I739008(cI) , BBa_I739009(ECFP) | reporting subsystem |
BBa_I739204 | kanamycin | low | BBa_I739006(cI) , BBa_I739007(P22 cII) , BBa_I739010(RFP) , 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 here. For a shorter explanation of how to assemble 2 parts together check 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. Composite parts made of parts a and b are denoted a.b.Plasmid 1 (BBa_I739201)
- Digest I739001(TetR) and BBa_I739201 plasmid with EcoRI/PstI and ligate them afterwards.
- Digest I739002(LacI) and I739003(LuxR) with XbaI/PstI
- Digest I739001(TetR) in BBa_I739201 with SpeI/PstI.
- Ligate digested I739001(TetR) in BBa_I739201 with digested I739002(LacI). You get a plasmid containing a I739001(TetR).I739002(LacI) composite part.
- Digest I739001(TetR).I739002(LacI) with SpeI/PstI.
- Ligate digested I739001(TetR).I739002(LacI) with digested I739003(LuxR). You get the completed I739001(TetR).I739002(LacI).I739003(LuxR) composite part in the BBa_I739201 plasmid.
Plasmid 2 (BBa_I739202)
- 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 (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] QIAGEN FAQs