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- | {{:Team:EPF-Lausanne/Templates/Header|title=Reporter plasmids assembly}}
| + | #REDIRECT [[Team:EPF-Lausanne/Our_Project/Reporter_Systems]] |
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- | We are building a system containing TetR and LacI, that control the expression of a selection gene. The sequential use of these two repressors allows us to have a direct '''activation''' of the selection gene when TetR '''binds''' its recognition sequence.
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- | [[File:EPFL_Summary_(with_TFs).png|650 px]]
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- | The selection gene is either RFP or a lysis cassette. RFP allows direct recognition of correctly assembled plasmids, which is also useful to see if the transformation has been efficient. Furthermore, it is an easy way to detect TetR-Ptet interactions. The lysis cassette allows recovering DNA from mutants in which TetR binds to a sequence (either consensus sequence or modified one. This could be used in a chemostat device for a high-throughput <i>in vivo</i> selection.
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- | We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:
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- | * The '''Inverter plasmid''' containing TetR under a constitutive promoter and a LacI inverter (i.e. with a Ptet promoter)
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- | * The '''Reporter plasmid''' containing a reporter gene, either RFP or a lysis device
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- | [[File:TetR_and_2_reporters.jpg]]
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- | TODO: describe here the sequence of assembly for our plasmids: what plasmids we build; where the parts came from;
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- | what had to be done. For example, why did we have to rebuild J61002 from scratch?
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- | This page will probably become very technical, in which case we will move it and replace by a succinct outline.
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- | The goal of this page is to keep everybody on the assembly team up to date on the ugly details of everybody's work.
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- | Leave nothing out!
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- | == Reporter Plasmids ==
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- | The reporter plasmids contain a selection gene under LacI regulation, to be coupled with the Inverter plasmid carrying TetR and LacI. In presence of TetR the reporter gene is ''activated''. Two reporter plasmids are built: one that contains and RFP and one that contains a lysis gene (also called lysis plasmid) to lyse the cells that contain valid combinations of TetR and pTet mutants, in order to recover their DNA. Their structure is illustrated below.
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- | [[File:EPFL-Reporter_plasmids_both.png|700px|center]]
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- | === Backbone template assembly ===
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- | The '''first step''' of assembly was to make a J61002 backbone, containing pTet and RFP. We took the J61002 plasmid from the registry, containing ampicillin resistance, RFP and a ColE1 replication origin (medium copy number). By PCR, we then added the pTet promoter in front of RFP. Finally, the pTet-RFP and the backbone (plasmid without RFP) were assembled thank to Gibson method. This was our first successful Gibson assembly.
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- | {|
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- | ! Part used for Gibson assembly
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- | ! Amplified from which template
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- | ! Features added with the PCR primers
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- | |-
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- | | J61002 backbone
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- | | J61002 plasmid - from delivery kit
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- | | Terminator B0014
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- | | Ptet-RFP
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- | | J61002 plasmid - from delivery kit
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- | | Ptet, RBS and spacer
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- | |}
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- | [[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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- | This plasmid can also be used as a reporter plasmid when transformed in combination with the TetR plasmid (pSB3K1 Pconst-TetR); RFP is then induced by ATC (anhydritetracycline). It was actually used for testing our mutants TetR in vivo.
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- | [[File:EPFL_TetR_and_Ptet-RFP.jpg|600px]]
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- | === Adding the reporter ===
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- | The '''second step''' is to add the reporter gene, either RFP or lysis cassette, under Plac promoter in the J61002 backbone. Let's run through the elements one by one. We need:
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- | * The '''backbone''', containing the replication origin and Ampicillin resistance
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- | * a '''pLac promoter'''...
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- | * ...that represses expression of a '''reporter gene''' either RFP or the Lysis cassette.
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- | TODO: - illustrate which parts are copied from where.
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- | - lookup biobrick numbers.
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- | The '''backbone''' is copied from the previously-assembled J61002-pTet-RFP. The RFP gene is '''not''' included in this copy; this allows the same PCR primers and products to be used for the assembly of both plasmids. In the case of the '''RFP''' plasmid, RFP is copied from the J61002-pTet-RFP plasmid, making a fragment separate from the backbone. In the case of the '''Lysis''' plasmid, the lysis device is copied from the T4 Lysis device plasmid, from the iGEM gene distribution. In both cases, the '''Plac''' promoter is added in the primer sequence.
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- | {|
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- | ! Part used for Gibson assembly
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- | ! Amplified from which template
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- | ! Features added with the PCR primers
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- | |-
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- | | J61002 backbone
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- | | J61002 pTet-RFP plasmid - from previous assembly
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- | | none
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- | | Plac-RFP
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- | | J61002 pTet-RFP plasmid - from previous assembly
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- | | Plac (already containing RBS and spacer)
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- | | Plac-lysis
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- | | T4 lysis plasmid
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- | | Plac (already containing RBS and spacer)
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- | |}
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- | [[File:EPFL_Reporter_plasmids_1ststep_both.jpg|680px]]
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- | The resulting plasmids express either RFP or the lysis genes under Plac control. For the '''lysis''' plasmid, we transformed the Gibson-assembled plasmids into Bl21 E.coli strain. They constitutively express LacI, which represses the lysis cassette and allow the correct mutants to grow.For the '''RFP''' plasmid -and all the other assemblies- we used the DH5alpha strain.
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- | == TetR plasmid ==
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- | === Inserting TetR under constitutive promoter ===
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- | This assembly was quite tricky, as we had to test 3 plasmids before succeeding.
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- | * We first took the J23019 plasmid from the registry and designed plasmids, but we never succeeded in amplifying it during PCRs. We assumed that probably the plasmid is not exactly the one described in the registry and went on with plasmid number 2.
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- | * Our second choice was pSB3C5, as we could partially reuse the primers designed for J23019. But the PCR results were very weak and the assembly failed.
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- | * So finally we used '''pSB3K1''', which is really similar to pSB3C5, designed new primers and succeeded in doing the Gibson assembly!
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- | We amplified this backbone, and added '''TetR''' under '''constitutive promoter''' to it.
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- | {|
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- | ! Part used for Gibson assembly
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- | ! Amplified from which template
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- | ! Features added with the PCR primers
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- | | pSB3K1 backbone
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- | | pSB3K1 plasmid - from registry
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- | | none
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- | | Pconst-TetR
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- | | Repressilator plasmid
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- | | Constitutive promoter (Pconst), RBS and spacer
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- | |}
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- | [[File:EPFL_Tetr_plasmid.jpg|300px]]
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- | === 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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- | [[File:EPFL_TetR_plasmid_cut_out_RFP.jpg|680px]]
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- | === Adding Ptet-LacI ===
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- | In order to test our lysis device, we added '''LacI''' under Ptet promoter to '''pSB3K1 Pconst-TetR''' (after RFP has been cut out).
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- | {|
| + | |
- | ! Part used for Gibson assembly
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- | ! Amplified from which template
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- | ! Features added with the PCR primers
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- | |-
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- | | pSB3K1 Pconst-TetR backbone
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- | | pSB3K1 Pconst-TetR plasmid - from previous digestion
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- | | Terminator B0014
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- | |-
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- | | Ptet-LacI
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- | | Repressilator plasmid
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- | | Ptet, RBS and spacer
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- | |}
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- | [[File:EPFL_TetR_plasmid_with_LacI.jpg]]
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- | {{:Team:EPF-Lausanne/Templates/Footer}}
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