From 2007.igem.org

In this page, you may found information on 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 an (unfortunately not complete) electronic copy of our lab notebook. If you are here because you are interested in implementing educatethe.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):]

• 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:]

• 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	pBR322 Map
pBR322	Cloned in linker oligos (EcoRI/PstI)	BBa_I739203	Tetracycline
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
pACYC177	Cloned in linker oligos (BamHI/PstI)	BBa_I739204	Kanamycin	pACYC177 Map

Cloning plan

Parts assignment into plasmids

Three plasmids are used for the EducatETH E.coli system parts as follows:

Plasmids and contents

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:

Linkers for plasmids

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)

1. Put parts 1,2,3 in pbr322ap plasmids.
2. Merge plasmid containing part 2 (donor) with plasmid containing part 3 (acceptor). You should get a plasmid containing a 2.3 composite part.
3. 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)

1. Put parts 4,5,8,9 in pck01cm plasmids.
2. Merge plasmid containing part 4 (donor) with plasmid containing part 5 (acceptor). You should get a plasmid containing a 4.5 composite part.
3. 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.
4. 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)

1. Put parts 6,7,10,11 in pacyc177km plasmids.
2. Merge plasmid containing part 6 (donor) with plasmid containing part 7 (acceptor). You should get a plasmid containing a 6.7 composite part.
3. 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.
4. 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.

Labbook

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]