Team:Birkbeck/Composite Part

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Composite Parts

tfa (tail fibre assembly) gene circuit (BBa_K1846001)

The tfa (tail fibre assembly) protein of bacteriophage Lambda assists in the assembly of the stf (short tail fibre) protein into a functional tail fibre. This part provides the gene sequence for tfa, together with a TetR repressible promoter (TetO), ribosome binding site and an rrNB T1 terminator. Presence of the gene in shipping backbone pSB1C3 was confirmed by restriction with enzymes EcoRV and PstI (Fig. 1) and by Sanger sequencing.

a.      b. 

Fig 1. a. Predicted band size of correct (lane 1) and incorrect (lane 2) restriction. b. Observed band sizes of both samples (both the tfa circuit) match correct band sizes of 1133 and 1737 bp.

TetR circuit (BBa_K1846003)

This is a constitutive part creating a circuit for the production of the tetracyline repressor TetR, using a constitutive, medium-copy chloramphenicol promoter, ribosome binding sequence and double terminator (rrnb T1 terminator followed by a T7Te terminator).

Correct cloning of the part into the pSB1C3 shipping vector was confirmed by Sanger sequencing.

TetR-regulated tfa (tail fibre assembly) circuit (BBa_K1846007)

To control production of the tail fibre assembly protein to prevent toxicity to the cell, we have combined the TetR circuit (BBa_K1846003) and the tfa circuit (BBa_K1846001) into a single BioBrick. With the production of the tfa protein under control of a Tet-repressible promoter, the coding sequence will remain inactive due to production of TetR. However, on addition of anhydrous tetracycline, TetR will preferably bind to this molecule, allowing the initiation of transcription.

cI-Cro circuit (BBa_K1846005)

We have created a regulatory circuit to control the lysogenic and lytic phases of bacteriophage lambda. The cI-Cro construct contains a circuit for the production - via a constitutive promoter - of the cI repressor protein (also known as the Lambda Repressor), which is responsible for keeping bacteriophage lambda in the lysogenic cycle through the cooperative binding of two repressor dimers to the DNA, repressing the Cro gene. The circuit uses a T7 promoter to drive the expression of the Cro gene in the opposite direction to the cI gene, essentially silensing the expression of the cI gene. The strength of the promoter used means that in the presence of T7 DNA polymerase, production of the Cro repressor will incapacitate production of the cI repressor and thus enables the switch from lysogenic cycle to the lytic cycle.

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