ETHZ/Biology/Lab

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.:: EducatETH E. coli - Lab Notes ::.

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


Contents

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:

  • pBR322:













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 we faced might be useful to you. If you want to see the whole biological design of the system, please visit the Biology Pespective. Finally, photos of our lab experience are accessible under Pictures!

Todo: decide what happens with lab book (here)

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].
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. 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.

Problems we faced

Lab book

Week 1

Date TODO's Completed People
Mon, 06. Aug. 2007
  • Preparing the Solutions
Sylke
Raphael
Stefan
Markus
Martin
Christos
Joe
Tue, 07. Aug. 2007
  • Prepare competent cells for all parts
  • Transformation of all the parts
Sylke
Raphael
Stefan
Markus
Martin
Christos
Joe
Wed, 08. Aug. 2007
  • Preparing the grown cultures (12) for the MINIPREP
    (o/n cultures)
Raphael
Stefan
Thu, 09. Aug. 2007
  • MINIPREP of the grown (10) o/n cultures
  • Gelelectrophoresis of the grown cultures (step: 0.8% Agarose)
Raphael
Stefan
Martin
Christos
Joe
Fri, 10. Aug. 2007
  • 7 working parts/plasmids (step after "DIGESTS"):
    (B0034, R0062, R0053, E0434, B0015, R0010, E0422)
  • 4 parts/plasmids minipreped:
    (R0040, R0051, Q04121, C0053)

Christos
Markus
Stefan

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
  • Prepare competent cells
  • Transformations of J23100, J37033, Q04400, Q04510
  • Control Restrictions (step after "MINIPREP")
    R0040, R0051, Q04121, C0053
  • o/n culture (E.Coli Top10)
  • Control Restrictions (didn't work)
Martin
Markus
Christos
Tim
Tue, 14. Aug. 2007 Morning Shift:
  • Start Preparing competent cells (for J23100, J37033, Q04400, Q04510)

Evening Shift:

  • Transformations of J23100, J37033, Q04400, Q04510
Morning Shift:
  • Prepared competent cells (stored in the -80°C freezer in the basement)

Evening Shift:

  • Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday)
  • Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4%
Morning Shift (9am-1pm?):
Markus
Tim
Evening Shift (5pm-...):
Martin
Christos
Wed, 15. Aug. 2007
  • Ligation (step: "LINK ASSEMBLY"):
    R0053 + E0422
    R0010 + E0422
    R0010 + E0434
    S/P: R0053, R0010
    X/P: E0422, E0434
  • Ligation didn't work due to bad quality of enzymes (probably)
From 12:
Martin
Markus

Thu, 16. Aug. 2007
  • Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)
  • Transformation of #13 and #14
  • Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510)
    One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)
  • Transformation of #13 (Q04121) and #14 (C0053)
    Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)

Markus
Christos
(Martin)

Fri, 17. Aug. 2007
  • o/n of #13 and #14
  • Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful
  • #13 and #14 didn't grow
  • # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates
  • New white pipette tips prepared (autoclave)
  • New bottles of Liquid LB and LB Agar prepared (autoclave)
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
  • Transform pbr322, pcyc177 and pck01
  • Transform pbr322, pcyc177 and pck01 and plated them
Stefan
Sun, 02. Sept. 2007
  • Prepare o/n of pbr322, pcyc177, pck01
  • o/n of pcyc177, pck01
  • the plates of pcyc177 and pck01 are in the fridge
  • transformed pbr322 because the culture didn't grow on the plate
Stefan

Week 5

Date TODO's Completed People
Mon, 03. Sept. 2007
  • Prepare new competent cells
  • Miniprep pcyc177 and pcK01
  • prepare new o/n culture of pbr322
  • Run agarose gel of Minipreped plasmids
  • New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)
  • Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)
  • pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday
  • new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids
  • 2 boxes of blue pipette tips are in the autoclave
  • Stefan ran the agarose gel (?)
Martin
Stefan
Tue, 04. Sept. 2007
  • Miniprep pcyc177 and pck01
  • cut the prepped plasmids to test if we've got the right ones
  • run agarose gel to test the cut and uncut ones
  • prepare new o/n of pbr322 (from Andy)
  • Miniprep of pcyc177 and pck01 (but not yet tested)
  • Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep
Martin
Christian
Wed, 05. Sept. 2007
  • Miniprep of pbr322
  • Test-Digest of pcyc177 and pck01 and agarose gel...
  • Streak out all three plasmids on new plates, so we have them in reserve
  • New Plate of pbr322.
  • Minipreps and Agarose Gels will be done tomorrow
Martin
Thu, 06. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Test with agarose gel
  • Gel of the older plasmids -> plasmid present
Christian
Fri, 07. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Plasmids miniprepped
Martin
Sat, 08. Sept. 2007
Sun, 09. Sept. 2007

Week 6

Date TODO's Completed People
Mon, 10. Sept. 2007
  • Miniprep pBR322
  • annealing of different MCSs
  • Digest of pCK01 with BamHI+AseI
  • digest of pACYC177 with BamHI+PstI
  • digest of pBR322 with EcoRI+PstI 
 all digests o/n

Christian

Tue, 11. Sept. 2007
  • Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work
  • 1x ligation of MCS inside backbones o/d, Trafo
  • 1x ligation of MCS inside backbones o/n
  • plate all 3 plasmids for new minipreps

Christian

Wed, 12. Sept. 2007
  • Trafo of o/n ligations
  • o/n cultures of putative clones

Christian

Thu, 13. Sept. 2007
  • Minipreps of putative clones pCK01-MCS and pBR322-MCS
  • control digests of putative clones
  • new o/n cultures of the putative clones of o/n ligations

Christian

Fri, 14. Sept. 2007
  • separation of control digests of putative clones

*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
  • new digest of pACYC177 with BamHI+PstI o/n
  • digest of pACYC177, pBR322 AP
  • ligation of 177 and 322AP
  • digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)
  • ligation of pBR322 o/n
  • 100 ml o/n culture to MAXIprep pacyc177
  • Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)

Christian
Martin, Raphael

Tue, 18. Sept. 2007
  • different control digests of pBR322-MCS (Tet) (see last week)
  • separation of pACYC177 digest
  • Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)
  • Transformation of the ligated pbr322 AP (MCS)
  • Prep pacyc177
  • Digest prepped pacyc177

> no DNA on pACYC177 digest-gel, only degradation smear

  • Plates of pbr322 AP grew
  • No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)
  • Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )
  • Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.
  • Digest of pacyc177 with PstI o/n (pray that it will work!)
  • New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)
  • test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests

Christian
Martin
Raphael

Wed, 19. Sept. 2007
  • o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated
  • Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)
  • check the digests of pacyc177 (pst) and pck01 (notI)
  • design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01
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

References

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

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