Team:EPF-Lausanne/Our Project/Assembly/Assembly details

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[[File:EPFL-J61002-pTet-RFP.png|400px]]
[[File:EPFL-J61002-pTet-RFP.png|400px]]
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The product of this assembly is the J61002-pTet-RFP 'backbone' plasmid. It contains an ampicillin resistance gene, a middle copy-number p15A origin, as well as RFP repressed by Ptet. This plasmid is used as a template for the second step of assembly, in which the LacI inverter and reporter genes are introduced.
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The product of this assembly is the J61002-pTet-RFP 'backbone' plasmid. It contains an ampicillin resistance gene, a middle copy-number p15A origin, as well as RFP repressed by Ptet. You can access the complete sequence of the plasmid [https://static.igem.org/mediawiki/2011/f/f8/EPFL_J61002_ptet-RFP.txt here]. This plasmid is used as a template for the second step of assembly, in which the LacI inverter and reporter genes are introduced.
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The resulting plasmid is called the '''Reporter plasmid''', it is used in our [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_LacI_-_RFP_system second readout system] along with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#Second_plasmid_-_pSB3K1_Pconst-TetR_Ptet-LacI Inverter plasmid].
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The resulting plasmid is called the '''Reporter plasmid''', it is used in our [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_LacI_-_RFP_system second readout system] along with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#Second_plasmid_-_pSB3K1_Pconst-TetR_Ptet-LacI Inverter plasmid]. You can access the complete sequence of the plasmid on [https://static.igem.org/mediawiki/2011/4/42/EPFL_J61002_Plac-RFP.txt this page].
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== TetR plasmids ==
== TetR plasmids ==
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* Pconst: J23116 from Berkeley 2006 [http://partsregistry.org/Part:BBa_J23116 "J23116"] (sequence copied into our primers)
* Pconst: J23116 from Berkeley 2006 [http://partsregistry.org/Part:BBa_J23116 "J23116"] (sequence copied into our primers)
* RBS: B0034 from the Registry [http://partsregistry.org/Part:BBa_B0034 "B0034"] (sequence copied into our primers)
* RBS: B0034 from the Registry [http://partsregistry.org/Part:BBa_B0034 "B0034"] (sequence copied into our primers)
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* TetR: [https://static.igem.org/mediawiki/2011/b/b0/EPFL_TetR_sequence.txt "TetR sequence"] (623 bp) The sequence lacks a stop codon, so we added one (TAA) with our primers
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* TetR: [https://static.igem.org/mediawiki/2011/b/b0/EPFL_TetR_sequence.txt "TetR sequence"] (623 bp)- The sequence lacks a stop codon, so we added one (TAA) with our primers.
[[File:EPFL_Tetr_plasmid.jpg|300px]]
[[File:EPFL_Tetr_plasmid.jpg|300px]]
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Our '''TetR plasmid''' contains a p15A replication origin, the same medium-copy number as in J61002 to ensure that during cotransformations the 2 different plasmids are present in same amounts. The plasmids carries a Kanamycin resistance marker, also because of cotransformations with J61002: we need a different selection antibiotic for each plasmid.
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Our '''TetR plasmid''' contains a p15A replication origin, the same medium-copy number as in J61002 to ensure that during co-transformations the 2 different plasmids are present in same amounts. The plasmid carries a Kanamycin resistance marker, since co-transformations with J61002 means we need a different selection antibiotic for each plasmid to confirm both are present (J61002 = Ampicillin resistance).
=== Cutting out RFP ===
=== Cutting out RFP ===
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We were surprised to see that the colonies recovered after Gibson assembly were red, indicating that the pSB3K1 plasmid contains RFP. By looking at it more carefully, we discovered that we had included the Biobrick region with our primers, which explains why RFP was present in our pSB3K1 backbone. taking advantage of the BioBrick format, we digested our pSB3K1 Pconst-TetR plasmid with SpeI and XbaI and then successfully religated.
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We were surprised to see that the colonies recovered after Gibson assembly were red, indicating that the pSB3K1 plasmid contains RFP. By looking at it more carefully, we discovered that we had included the Biobrick region with our primers, which explains why RFP was present in our pSB3K1 backbone. Taking advantage of the BioBrick format, we digested our pSB3K1 Pconst-TetR plasmid with SpeI and XbaI and then successfully religated.
[[File:EPFL_TetR_plasmid_cut_out_RFP.jpg|680px]]
[[File:EPFL_TetR_plasmid_cut_out_RFP.jpg|680px]]
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Now we have the expected ''TetR plasmid'' that is used in combination with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#First_reporter_-_J61002_Ptet-RFP J61002 Ptet-RFP plasmid] into our [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_RFP_system first readout system].
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Now we have the expected ''TetR plasmid'' that is used in combination with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#First_reporter_-_J61002_Ptet-RFP J61002 Ptet-RFP plasmid] into our [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_RFP_system first readout system]. The complete sequence of the plasmid is available[https://static.igem.org/mediawiki/2011/5/5a/EPFL_PSB3K1_Pconst-TetR.txt here].
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Complete sequence of the plasmid: [https://static.igem.org/mediawiki/2011/5/5a/EPFL_PSB3K1_Pconst-TetR.txt "pSb3K1 Pconst-TetR"]
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This plasmid was successfully sequence-verified:
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*Sequencing data compared to the sequence of Pconst,RBS+spacer and TetR gene:[https://static.igem.org/mediawiki/2011/2/23/EPFL_pSb3K1_TetR_seq.txt "pSb3K1_TetR_seq"]
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=== Second plasmid - pSB3K1 Pconst-TetR Ptet-LacI ===
=== Second plasmid - pSB3K1 Pconst-TetR Ptet-LacI ===
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Details of the parts assembled:
Details of the parts assembled:
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* Plasmid backbone containing Pconst and TetR: see precedent section
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* Plasmid backbone containing Pconst and TetR: see preceding section
* Terminator: B0014 from the Registry [http://partsregistry.org/Part:BBa_B0014 "B0014"] (sequence copied into our primers)
* Terminator: B0014 from the Registry [http://partsregistry.org/Part:BBa_B0014 "B0014"] (sequence copied into our primers)
* Ptet: R0040 from Registry [http://partsregistry.org/Part:BBa_R0040 "R0040"] (sequence copied into our primers)
* Ptet: R0040 from Registry [http://partsregistry.org/Part:BBa_R0040 "R0040"] (sequence copied into our primers)
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* LacI: amplified from Repressilator plasmid [https://static.igem.org/mediawiki/2011/7/7f/EPFL_LacI_sequence.txt "LacI sequence"] The sequence lacks a stop codon, we added TAA with our primers.
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* LacI: amplified from Repressilator plasmid [https://static.igem.org/mediawiki/2011/7/7f/EPFL_LacI_sequence.txt "LacI sequence"]- The sequence lacks a stop codon, so we added one (TAA) with our primers.
[[File:EPFL_TetR_plasmid_with_LacI.jpg]]
[[File:EPFL_TetR_plasmid_with_LacI.jpg]]
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This '''Inverter plasmid''' now has a cascade reaction consisting of TetR constitutively expressed that represses LacI. It still contains p15A origin and a kanamycin resistance marker, being compatible with the J61002 plasmids for cotransformations.
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This '''Inverter plasmid''' now contains a cascade reaction consisting of TetR constitutively expressed that represses LacI. It still contains p15A origin and a kanamycin resistance marker, making it compatible for cotransformations with the J61002 plasmids.
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The '''Inverter palsmid''' is used in combination with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#Second_reporter_-_J61002_Plac-RFP Reporter plasmid] in our second reporter system. To see the experimental results of this system, please go on [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_LacI_-_RFP_system this] page.
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The '''Inverter plasmid''' is used in combination with the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly/Assembly_details#Second_reporter_-_J61002_Plac-RFP Reporter plasmid] in our second reporter system. To see the experimental results of this system, please go on [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_LacI_-_RFP_system this] page.

Latest revision as of 16:58, 21 September 2011