ETHZ/Biology/Lab

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<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br>
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<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - Lab Notes ::. </b></font></center><br>
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<div class="ddcolortabsline">&nbsp;</div>
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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 [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]
 
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Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])
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<div id="dropmenu_home" class="dropmenudiv_a">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Team_Members">Team Members</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Acknowledgments">Acknowledgments</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Site_Map">Site map</a>
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</div>
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==Cloning plan==
 
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===Parts assignment into plasmids===
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<div id="dropmenu_modeling" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Model_Overview">Model Overview</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Detailed_Model">Detailed Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Final_Model">Final Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Modeling_Basics">Modeling Basics Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Mathematical_Model">Mathematical Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/FSM">FSM View Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/FlipFlop">Flip-Flop View Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Parameters">Parameters Page</a>
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Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:
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<div id="dropmenu_simulation" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Simulation_of_Test_Cases">Test Cases</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Sensitivity_Analysis">Sensitivity Analysis</a>
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</div>
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|+'''Plasmids and contents'''
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<div id="dropmenu_biology" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Introduction">Introduction</a>
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! plasmid !! resistance !! copy type!! contents !! comments
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#The_Complete_System">The Complete System</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#System_Phases">System Phases</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Current_Cloning_Status">Current Cloning Status</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology/parts">System Parts Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology/Lab">Lab Notes Page</a>
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| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#The_ETH_Zurich_07_Team">The ETH Zurich 07 Team</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#Team_Description">Team Description</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Internal">Brainstorming Page</a>
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| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem
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| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem
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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.
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= Introduction =
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===Linkers===
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On this page, you can find information about how educatETH <i>E.coli</i> 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, you can find [[ETHZ/Biology/Labbook| here]] an (unfortunately not complete) electronic copy of our lab notebook. If you are here because you are interested in implementing educatETH <i>E.coli</i> in your lab, then our System Implementation and the System Parts pages may be of help to you!
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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:
+
For all our cloning procedures we used standard protocols according to ''SAMBROOK and RUSSELL'' Molecular Cloning: A Laboratory Manual [1].
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== Strains ==
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|+'''Linkers for plasmids'''
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|-
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! Linker!! Plasmid
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| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322
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We used the following <i>E. coli </i> strains:
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|-
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| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322
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|-
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| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322
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|-
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| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322
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|-
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| [[ETHZ/pck01| pck01]] || pck01
 
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|-
 
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| [[ETHZ/pCK01-2| pck01-2]] || pck01
 
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|-
 
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| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177
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[http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29|<b><i>E. coli </i>Top10 (Invitrogen):</b>] <br>
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|-
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*You can find this strain at [http://partsregistry.org/Part:BBa_V1009 BBa_V1009]
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| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177
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*This strain has a streptomycin resistance <br>
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|-
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*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
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|}
+
*For further information please [http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29| click here]
 +
*<i>References</i>:
 +
**Casdaban, M. and Cohen, S. (1980) J Mol Biol 138:179 PMID 6997493 <br>
 +
**Grant, S.G.N. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 4645-4649 PMID 2162051
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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.
 
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=== Procedure ===
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[http://openwetware.org/wiki/E._coli_genotypes#JM101|<b><i>E. coli </i>JM101:</b>] <br>
 +
*You can find this strain at [http://partsregistry.org/Part:BBa_I739301 BBa_I739301]
 +
*We call them <i>Jimmys</i>
 +
*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]
 +
*<i>Reference</i>:
 +
**Messing, J. et al. (1981) Nucleic Acids Res. 9, 309; Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103
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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]. [[Image:Assembly _process.png|thumb|300px|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'''.
 
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==== Plasmid 1 ''(pbr322ap)'' ====
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== Plasmids ==
 +
For our system we needed three plasmids with different origins of replication and antibiotic resistances. We decided to take low copy plasmids. We also decided to use the following plasmids, which we wanted to modify so that they would become compatible to the Biobrick Library multiple cloning site:
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# Put parts 1,2,3 in  pbr322ap plasmids.
 
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# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.
 
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# 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.
 
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==== Plasmid 2 ''(pck01cm)''====
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=== Basic plasmids ===
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# Put parts 4,5,8,9 in  pck01cm plasmids.  
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# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.
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|-
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# 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.
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! Plasmid !! Resistances !! Copy number !! Origin !! Map
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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.
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|-
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| [[ETHZ/pbr322| pBR322]] [2,3] || width=505px | Ampicillin, Tetracyline || 15-20 [4] || width=96px | pMB1 || [[Image:Mappbr322.jpg|center|thumb|pBR322 Map|100px]]
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|-
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| [[ETHZ/pck01| pCK01]] [5] || Chloramphenicol|| 5-12 [4] || pSC101 || [[Image:Mappck01.jpg|center|thumb|pCK01 Map|100px]]
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|-
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| [[ETHZ/pacyc177| pACYC177]] [6,7] || Ampicillin, Kanamycin|| 10-12 [4] || p15A || [[Image:Mappacyc177.jpg|center|thumb|pACYC177 Map|100px]]
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|-
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|}
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<br>
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====Plasmid 3 ''(pacyc177km)''====
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=== 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:
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# Put parts 6,7,10,11 in  pacyc177km plasmids.
 
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# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.
 
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# 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.
 
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# 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.
 
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== Problems we faced==
 
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== Lab book ==
 
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==== Week 1 ====
 
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! Date                       
 
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! TODO's             
 
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! Completed             
 
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! People
 
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| Mon, 06. Aug. 2007         
 
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|
 
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|                   
 
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* Preparing the Solutions                     
 
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| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe
 
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|-
 
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| Tue, 07. Aug. 2007         
 
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|                   
 
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|
 
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* Prepare competent cells for all parts
 
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* Transformation of all the parts                     
 
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| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe
 
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|-
 
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| Wed, 08. Aug. 2007         
 
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|                   
 
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|
 
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* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures)                     
 
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| Raphael<br>Stefan
 
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|-
 
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| Thu, 09. Aug. 2007         
 
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|                   
 
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|
 
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* MINIPREP of the grown (10) o/n cultures
 
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* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose)                     
 
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| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe
 
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|-
 
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| Fri, 10. Aug. 2007         
 
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|                   
 
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|
 
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* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)
 
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* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)
 
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|
 
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Christos <br> Markus <br> Stefan
 
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|-
 
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| Sat, 11. Aug. 2007         
 
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|                   
 
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| no labwork                     
 
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| Sun, 12. Aug. 2007       
 
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| labwork cancelled
 
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==== Week 2 ====
 
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! Date                       
 
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! TODO's             
 
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! Completed             
 
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! People
 
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|-
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| Mon, 13. Aug. 2007 <br> start at 3 pm       
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! Plasmid !! Changes !! width=85px| New name !! New resistance !! New Map
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|
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* Prepare competent cells
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* Transformations of J23100, J37033, Q04400, Q04510
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* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053
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|                                 
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* o/n culture (E.Coli Top10)
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* Control Restrictions (didn't work)
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|  Martin<br> Markus <br> Christos <br> Tim <br>
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|-
|-
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| Tue, 14. Aug. 2007         
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| width=94px | [http://partsregistry.org/Part:BBa_I739201 BBa_I739201]
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| Morning Shift: <br>
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* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br>                   
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Evening Shift: <br>
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* Transformations of J23100, J37033, Q04400, Q04510
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| Morning Shift: <br>
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* Prepared competent cells (stored in the -80°C freezer in the basement)    <br>
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Evening Shift: <br>
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* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday)
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* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4%                   
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|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br>  Christos
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|-
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| Wed, 15. Aug. 2007         
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|   
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* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434
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|
|
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* Ligation didn't work due to bad quality of enzymes (probably)                       
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*Site directed mutagenesis: Changed the GCA codon of the PstI site of the bla gene into GTA
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| From 12:<br> Martin<br>Markus<br><br>
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*Cloned in [[ETHZ/pbr322-3| linker oligos]] (EcoRI/BamHI)
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|-
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| Thu, 16. Aug. 2007         
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|                   
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* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)
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* Transformation of #13 and #14
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|                       
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* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> 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)
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* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)
+
|
|
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Markus<br>Christos<br>(Martin)
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[[ETHZ/pbr322| pBR322]]
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|
 +
Ampicillin
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|
 +
[[Image:MapI739201.jpg|center|thumb|BBa_I739201 Map|100px]]
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|-
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| Fri, 17. Aug. 2007         
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| [http://partsregistry.org/Part:BBa_I739203 BBa_I739203]
|
|
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* o/n of #13 and #14
+
*Cloned in [[ETHZ/pbr322-1| linker oligos]] (EcoRI/PstI)
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* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful
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|
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|  
+
[[ETHZ/pbr322| pBR322]]
-
* #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
+
Tetracycline
-
* New white pipette tips prepared (autoclave)
+
|
-
* New bottles of Liquid LB and LB Agar prepared (autoclave)
+
[[Image:MapI739203.jpg|center|thumb|BBa_I739203 Map|100px]]
-
| Martin
+
|-
|-
-
| Sat, 18. Aug. 2007         
+
| [http://partsregistry.org/Part:BBa_I739202 BBa_I739202]
-
|                    
+
|
-
|                      
+
*[[ETHZ/primer_pcr_killspe_up| Site directed mutagenesis]]: Changed the ACT codon of the SpeI site in the origin of replication into ATT
 +
*Cloned in [[ETHZ/pck01-3| linker oligos]] (AgeI/AseI)
 +
|
 +
[[ETHZ/pck01| pCK01]]
|
|
 +
Chloramphenicol
 +
|
 +
[[Image:MapI739202.jpg|center|thumb|BBa_I739202 Map|100px]]
|-
|-
-
| Sun, 19. Aug. 2007         
+
| [http://partsregistry.org/Part:BBa_I739204 BBa_I739204]
|
|
 +
*Cloned in [[ETHZ/pacyc177-1| linker oligos]] (BamHI/PstI)
|
|
 +
[[ETHZ/pacyc177| pACYC177]]
|
|
 +
Kanamycin
 +
| [[Image:MapI739204.jpg|center|thumb|BBa_I739204 Map|100px]]
 +
|-
|}
|}
 +
<br>
-
==== Week 3 ====
+
==Cloning plan==
-
Little rearrangements of the parts. Planning of the sequences to order them.
 
-
 
+
===Parts assignment into plasmids===
-
==== Week 4 ====
+
The plan was to put the following parts into the three plasmids (for the detailed cloning procedure see below):
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
-
! Date                       
+
|-
-
! TODO's             
+
! plasmid !! resistance !! copy type!! contents !! comments
-
! 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 ====
+
| [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
 +
|-
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+
| [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
-
! 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<br>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<br>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 ====
+
| [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
 +
|-
 +
|}
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+
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.
-
! 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 ====
 
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+
=== Cloning procedure ===
-
  ! Date                       
+
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]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process [8]]] 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'''.
-
! 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 <br> 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<br> <br>
+
-
* 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 <br>
 
-
Martin<br>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 ====
+
==== Plasmid 1 ''([http://partsregistry.org/Part:BBa_I739201 BBa_I739201])'' ====
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 
-
! 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 ====
+
# Digest '''[http://partsregistry.org/Part:BBa_I739001 I739001(TetR)]''' and [http://partsregistry.org/Part:BBa_I739201 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 [http://partsregistry.org/Part:BBa_I739201 BBa_I739201] with SpeI/PstI.
 +
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739001 I739001(TetR)]''' in [http://partsregistry.org/Part:BBa_I739201 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 [http://partsregistry.org/Part:BBa_I739201 BBa_I739201] plasmid.
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 
-
! 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 ====
 
-
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
+
==== Plasmid 2 ''([http://partsregistry.org/Part:BBa_I739202 BBa_I739202])''====
-
! Date                       
+
 
-
! TODO's             
+
 
-
! Completed             
+
# Digest '''[http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)]''', '''[http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)]''' and the plasmid [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with EcoRI/PstI.
-
! People
+
# 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 [http://partsregistry.org/Part:BBa_I739202 BBa_I739202].
-
| Mon, 8. Oct. 2007         
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)]''' and the plasmid [http://partsregistry.org/Part:BBa_I739202 BBa_I739202].
-
|
+
# Digest '''[http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)]''' in [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with SpeI/PstI.
-
|
+
# Digest '''[http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)]''' in [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with SpeI/PstI.
-
|
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)]''' in [http://partsregistry.org/Part:BBa_I739202 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 [http://partsregistry.org/Part:BBa_I739202 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.
-
| Tue, 9. Oct. 2007         
+
# 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.
-
|
+
 
-
|-
+
 
-
| Wed, 10. Oct. 2007           
+
 
-
|         
+
====Plasmid 3 ''([http://partsregistry.org/Part:BBa_I739204 BBa_I739204])''====
-
|
+
 
-
|
+
 
-
|-
+
# Digest '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)]''', '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)]''' and the plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with EcoRI/PstI.
-
| Thu, 11. Oct. 2007         
+
# Digest '''[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]''' and '''[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]''' with XbaI/PstI.
-
|
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)]''' and digested plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204].
-
|
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)]''' and the plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204].
-
|
+
# Digest '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)]''' in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with SpeI/PstI.
-
|-
+
# Digest '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)]''' in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with SpeI/PstI.
-
| Fri, 12. Oct. 2007         
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)]''' in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with digested '''[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]'''. You get a plasmid containing a '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]''' composite part.
-
|                   
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)]''' in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with '''[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]'''. You get a plasmid containing a '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]''' composite part.
-
|
+
# Digest '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]''' with SpeI/PstI.
-
|
+
# Digest '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]''' with XbaI/PstI.
-
|-
+
# Ligate digested '''[http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]''' and digested '''[http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]'''. You get the completed plasmid containing the '''[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)]''' composite part.
-
| Sat, 13. Oct. 2007           
+
<br>
-
|                   
+
-
|                       
+
-
|
+
-
|-
+
-
| Sun, 14. Oct. 2007       
+
-
|
+
-
|
+
-
|
+
-
|}
+
==References==
==References==
-
<p>[1] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]</p>
+
 
 +
[http://www.MolecularCloning.com &#91;1&#93; Sambrook J and Russel DW] <i>"Molecular Cloning: A Laboratory Manual"</i>, Cold Spring Harbour Laboratory Press, 3rd edition, 2001<br />
 +
&#91;2&#93; Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL and Boyer HW <i>"Construction of useful cloning vectors"</i>, Gene 2: 95-113, 1977<br />
 +
&#91;3&#93; Watson N <i>"A new revision of the sequence of plasmid pBR322"</i>, Gene 70: 399-403, 1988<br /> 
 +
[http://www1.qiagen.com/faq/faqview.aspx?faqid=350&SearchText=&FaqCategoryId=0&MenuItemId=0&catalog=1&ProductLineId=1000228  &#91;4&#93; QIAGEN FAQs]<br />
 +
[http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2958.1995.tb02293.x &#91;5&#93; Fernández S et al.] <i>"Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains"</i>, Mol Microbiol 16(2):205-213, 1995]<br />
 +
&#91;6&#93; Chang ACY and Cohen SN <i>"Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid"</i>, J Bacteriol 134: 1141-1156, 1978<br />
 +
&#91;7&#93; Rose, R.E., <i>"The nucleotide sequence of pACYC177"</i>, Nucleic Acids Res, 16(1): 356, 1988<br />
 +
[http://partsregistry.org/Assembly:Standard_assembly &#91;8&#93; Standard Assembly Process]<br />

Latest revision as of 20:03, 26 October 2007

ETHZ banner.png

 


Introduction

On this page, you can 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, you can 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!

For all our cloning procedures we used standard protocols according to SAMBROOK and RUSSELL Molecular Cloning: A Laboratory Manual [1].

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 also decided to use the following plasmids, which we wanted to modify so that they would become compatible to the Biobrick Library multiple cloning site:


Basic plasmids

Plasmid Resistances Copy number Origin Map
pBR322 [2,3] Ampicillin, Tetracyline 15-20 [4] pMB1
pBR322 Map
pCK01 [5] Chloramphenicol 5-12 [4] pSC101
pCK01 Map
pACYC177 [6,7] Ampicillin, Kanamycin 10-12 [4] 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
[http://partsregistry.org/Part:BBa_I739201 BBa_I739201]
  • Site directed mutagenesis: Changed the GCA codon of the PstI site of the bla gene into GTA
  • Cloned in linker oligos (EcoRI/BamHI)

pBR322

Ampicillin

BBa_I739201 Map
[http://partsregistry.org/Part:BBa_I739203 BBa_I739203]

pBR322

Tetracycline

BBa_I739203 Map
[http://partsregistry.org/Part:BBa_I739202 BBa_I739202]

pCK01

Chloramphenicol

BBa_I739202 Map
[http://partsregistry.org/Part:BBa_I739204 BBa_I739204]

pACYC177

Kanamycin

BBa_I739204 Map


Cloning plan

Parts assignment into plasmids

The plan was to put the following parts into the three plasmids (for the detailed cloning procedure see below):

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.


Cloning 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 [8]
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 ([http://partsregistry.org/Part:BBa_I739201 BBa_I739201])

  1. Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] and [http://partsregistry.org/Part:BBa_I739201 BBa_I739201] plasmid with EcoRI/PstI and ligate them afterwards.
  2. Digest [http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] and [http://partsregistry.org/Part:BBa_I739003 I739003(LuxR)] with XbaI/PstI
  3. Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] in [http://partsregistry.org/Part:BBa_I739201 BBa_I739201] with SpeI/PstI.
  4. Ligate digested [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)] in [http://partsregistry.org/Part:BBa_I739201 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.
  5. Digest [http://partsregistry.org/Part:BBa_I739001 I739001(TetR)].[http://partsregistry.org/Part:BBa_I739002 I739002(LacI)] with SpeI/PstI.
  6. 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 [http://partsregistry.org/Part:BBa_I739201 BBa_I739201] plasmid.


Plasmid 2 ([http://partsregistry.org/Part:BBa_I739202 BBa_I739202])

  1. Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)], [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] and the plasmid [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with EcoRI/PstI.
  2. Digest [http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] and [http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] with XbaI/PstI.
  3. Ligate digested [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] and digested plasmid [http://partsregistry.org/Part:BBa_I739202 BBa_I739202].
  4. Ligate digested [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] and the plasmid [http://partsregistry.org/Part:BBa_I739202 BBa_I739202].
  5. Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] in [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with SpeI/PstI.
  6. Digest [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] in [http://partsregistry.org/Part:BBa_I739202 BBa_I739202] with SpeI/PstI.
  7. Ligate digested [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)] in [http://partsregistry.org/Part:BBa_I739202 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.
  8. Ligate digested [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)] in [http://partsregistry.org/Part:BBa_I739202 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.
  9. Digest [http://partsregistry.org/Part:BBa_I739004 BBa_I739004(P22 cII)].[http://partsregistry.org/Part:BBa_E0430 BBa_E0430(EYFP)] with SpeI/PstI.
  10. Digest [http://partsregistry.org/Part:BBa_I739008 BBa_I739008(cI)].[http://partsregistry.org/Part:BBa_I739009 BBa_I739009(ECFP)] with XbaI/PstI.
  11. 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 ([http://partsregistry.org/Part:BBa_I739204 BBa_I739204])

  1. Digest [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)], [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)] and the plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with EcoRI/PstI.
  2. Digest [http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)] and [http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)] with XbaI/PstI.
  3. Ligate digested [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)] and digested plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204].
  4. Ligate digested [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)] and the plasmid [http://partsregistry.org/Part:BBa_I739204 BBa_I739204].
  5. Digest [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)] in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with SpeI/PstI.
  6. Digest [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)] in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with SpeI/PstI.
  7. Ligate digested [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)] in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with digested [http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)]. You get a plasmid containing a [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)] composite part.
  8. Ligate digested [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)] in [http://partsregistry.org/Part:BBa_I739204 BBa_I739204] with [http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]. You get a plasmid containing a [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)] composite part.
  9. Digest [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)] with SpeI/PstI.
  10. Digest [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)] with XbaI/PstI.
  11. Ligate digested [http://partsregistry.org/Part:BBa_I739006 BBa_I739006(cI)].[http://partsregistry.org/Part:BBa_I739007 BBa_I739007(P22 cII)] and digested [http://partsregistry.org/Part:BBa_I739010 BBa_I739010(RFP)].[http://partsregistry.org/Part:BBa_I739011 BBa_I739011(GFP)]. You get the completed plasmid containing the [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)] composite part.


References

[http://www.MolecularCloning.com [1] Sambrook J and Russel DW] "Molecular Cloning: A Laboratory Manual", Cold Spring Harbour Laboratory Press, 3rd edition, 2001
[2] Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL and Boyer HW "Construction of useful cloning vectors", Gene 2: 95-113, 1977
[3] Watson N "A new revision of the sequence of plasmid pBR322", Gene 70: 399-403, 1988
[http://www1.qiagen.com/faq/faqview.aspx?faqid=350&SearchText=&FaqCategoryId=0&MenuItemId=0&catalog=1&ProductLineId=1000228 [4] QIAGEN FAQs]
[http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2958.1995.tb02293.x [5] Fernández S et al.] "Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains", Mol Microbiol 16(2):205-213, 1995]
[6] Chang ACY and Cohen SN "Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid", J Bacteriol 134: 1141-1156, 1978
[7] Rose, R.E., "The nucleotide sequence of pACYC177", Nucleic Acids Res, 16(1): 356, 1988
[http://partsregistry.org/Assembly:Standard_assembly [8] Standard Assembly Process]

Retrieved from "http://2007.igem.org/wiki/index.php/ETHZ/Biology/Lab"