• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

Reporter plasmids assembly

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.

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 in vivo selection.

We need several plasmids to build our entire system. We chose a 2-plasmid strategy that looks like this:

• The Inverter plasmid containing TetR under a constitutive promoter and a LacI inverter (i.e. with a Ptet promoter)
• The Reporter plasmid containing a reporter gene, either RFP or a lysis device

Reporter Plasmids

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.

Backbone template assembly

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.

Details of the parts assembled:

• J61002 backbone: from Arkin lab [http://partsregistry.org/Part:BBa_J61002 "J61002"]
• Terminator B0014: from the Registry [http://partsregistry.org/Part:BBa_B0014 "B0014"] (sequence copied into our plasmids)
• Ptet: B0040 from the Registry [http://partsregistry.org/Part:BBa_R0040 "B0040"] (sequence copied into our plasmids)
• RBS: B0034 from the Registry [http://partsregistry.org/Part:BBa_B0034 2B00342] (sequence copied into our plasmids)
• RFP: sequence from J61002 plasmid [http://partsregistry.org/Part:BBa_J61002 "J61002"]

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.

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.

Adding the reporter

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:

• The backbone, containing the replication origin and Ampicillin resistance
• a pLac promoter...
• ...that represses expression of a reporter gene either RFP or the Lysis cassette.

TODO: - illustrate which parts are copied from where.
      - lookup biobrick numbers.

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 Reporter 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.

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.

TetR plasmid

Inserting TetR under constitutive promoter

This assembly was quite tricky, as we had to test 3 plasmids before succeeding.

• 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.
• 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.
• So finally we used pSB3K1, which is really similar to pSB3C5, designed new primers and succeeded in doing the Gibson assembly!

We amplified this backbone, and added TetR under constitutive promoter to it.

Details of the parts assembled:

• Plasmid backbone: pSB3K1 from ETHZ 2007 [http://partsregistry.org/wiki/index.php?title=Part:pSB3K1 "pSB3K1"] (taken from the delivery plate)
• Pconst: J23116 from Berkeley 2006 [http://partsregistry.org/Part:BBa_J23116 "j23116"] (sequence copied into our primers)
• RBS (B0034?) and spacer: (sequence copied into our primers)
• TetR: "TetR sequence" (623 bp) The sequence lacks a stop codon, we added TAA with our primers

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. We chose a constitutive promoter that was intermediarily strong, in order to have more repression of the lysis cassette by LacI in the Lysis-Inverter cotransformation, giving a better chance of surviving to the cells.

Cutting out RFP

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.

Now we have the expected TetR plasmid and we can go on with our assembly strategy.

Complete sequence of the plasmid: "pSb3K1 Pconst-TetR"

This plasmid was successfully sequence-verified:

• Sequencing data compared to the sequence of Pconst,RBS+spacer and TetR gene:"pSb3K1_TetR_seq"

Adding Ptet-LacI (Inverter plasmid)

In order to test our lysis device, we added LacI under Ptet promoter to pSB3K1 Pconst-TetR (after RFP had been cut out).

Details of the parts assembled:

• Plasmid backbone containing Pconst and TetR: see precedent section
• 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)
• LacI: amplified from Repressilator plasmid "LacI sequence" The sequence lacks a stop codon, we added TAA with our primers.

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.

Sequence of the plasmid: This plasmid was sequence-verified. While Pconst, TetR and LacI were OK, Ptet has been mutated.