• 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 built two different systems containing TetR and RFP, or TetR, LacI and RFP. TetR and LacI that control the expression of our RFP selection gene.

In the first readout system, RFP is repressed when TetR binds to its Ptet recognition sequence. However, the sequential use of two repressors allows us to have a direct activation of the selection gene when TetR binds its recognition sequence in our second readout system.

First readout system:

Second readout system:

We need several plasmids to build our two systems 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 RFP reporter gene

change image

Contents 1 Reporter Plasmids 1.1 First reporter - TetR 1.2 Second reporter - LacI 2 TetR plasmids 2.1 Inserting TetR under constitutive promoter 2.2 Cutting out RFP 2.3 Adding Ptet-LacI (Inverter plasmid)

Reporter Plasmids

The reporter plasmids contain RFP under TetR or LacI regulation, depending on which readout system they are used in. Their structure is illustrated below.

change image

First reporter - TetR

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.

Part used for Gibson assembly	Amplified from which template	Features added with the PCR primers
J61002 backbone	J61002 plasmid - from delivery kit	Terminator B0014
Ptet-RFP	J61002 plasmid - from delivery kit	Ptet, RBS and spacer

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 promoter: 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 gene: 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.

add sequence + seq results
add link to TetR plasmid

This plasmid is used in our first readout system, both with the wild-type TetR and with mutants. For the results of this readout system, please go on the [whatever] page.

add link

Second reporter - LacI

The second step is to add the RFP reporter gene 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 RFP reporter gene

The backbone is copied from the previously-assembled J61002-pTet-RFP. RFP is also copied from the J61002-pTet-RFP plasmid, making a fragment separate from the backbone. The Plac promoter is added in the primer sequence.

Part used for Gibson assembly	Amplified from which template	Features added with the PCR primers
J61002 backbone	J61002 pTet-RFP plasmid - from previous assembly	none
Plac-RFP	J61002 pTet-RFP plasmid - from previous assembly	Plac (already containing RBS and spacer)

Details of the parts assembled:

• J61002 backbone: from Arkin lab [http://partsregistry.org/Part:BBa_J61002 "J61002"]
• Plac promoter: new biobrick
• RFP gene: sequence from J61002 plasmid [http://partsregistry.org/Part:BBa_J61002 "J61002"]

put only RFP

The resulting plasmid is called the Reporter plasmid, it is used in our second readout system along with the Inverter plasmid.

add sequence + seq results
add link to Inverter
add link to results

TetR plasmids

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.

Part used for Gibson assembly	Amplified from which template	Features added with the PCR primers
pSB3K1 backbone	pSB3K1 plasmid - from registry	none
Pconst-TetR	Repressilator plasmid	Constitutive promoter (Pconst), RBS and spacer

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 from the Registry [http://partsregistry.org/Part:BBa_B0034 "B0034"] (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.

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 that is used in combination with the J61002 Ptet-RFP plasmid ino ur first readout system.

add link to J6 Ptet-RFP

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)

For the second readout system, we added LacI under Ptet promoter to pSB3K1 Pconst-TetR (after RFP had been cut out).

Part used for Gibson assembly	Amplified from which template	Features added with the PCR primers
pSB3K1 Pconst-TetR backbone	pSB3K1 Pconst-TetR plasmid - from previous digestion	Terminator B0014
Ptet-LacI	Repressilator plasmid	Ptet, RBS and spacer

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.

The Inverter palsmid is used in combination with the Reporter plasmid in our second reporter system. To see the experimental results of this system, please go on the [blabla] page.

add link to reporter palsmid
add link to 2nd system

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