• 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

Assembly Sequence

TODO: describe here the sequence of assembly for our plasmids: what plasmids we build; where the parts came from; 
what had to be done. For example, why did we have to rebuild J61002 from scratch?

This page will probably become very technical, in which case we will move it and replace by a succinct outline. 
The goal of this page is to keep everybody on the assembly team up to date on the ugly details of everybody's work.
Leave nothing out!

Reporter Plasmids

The reporter plasmid contains the mutated TetR operator coupled to a LacI inverter, so that in presence of TetR the reporter gene is activated. Two reporter plasmids are built: one that contains and RFP reporter 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.( vincent, please confirm this!). This was our first successful Gibson assembly.

Vincent, please document this step. What template did you use, what primers, etc? Please include links to the 
sequences of the template, of the expected product, and if possible a table that summarizes that all, 
like Nadine did.

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.

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, which already contains the pTet promoter (but we don't need it anymore...)
• 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.
      - illustrate Gibson overhangs

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 introduced with the primer.

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.

The Pst1 site is originally present in the J61002 plasmid, and was kept during the J61002 Ptet-RFP and Plac-RFP/lysis assemblies. We decided to use this restriction site as an alternative for producing linear fragments of the J61002 Plac-RFP/lysis plasmids. Instead of amplifying them by PCR, which may pose problem due to their size, we can miniprep them from cell cultures and then digest them.

Adding the inverter

The last step is to add the inverter, consisting of LacI under Ptet regulation. Here again we used Gibson assembly, but with a small trick: the backbone was really big, so we decided to cut it with a restriction enzyme instead of amplifying it by PCR. We took advantage of the PstI site; furthermore it is located just after a terminator, so we don't need to add one before Plac with our primers.

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.

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.

Adding Ptet-LacI

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