Team:Aachen/Parts

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iGEM Team Aachen BioBricks

This page lists the collection of BioBricks developed by our team for the project Cellock Holmes - A Case of Identity.


K1319000

RFC [25] Version of E0020

This part is an RFC [25]-compatible version of BBa_E0030. The start and stop codons have been removed to make it RFC [25]-compatible and the part is flanked by the RFC [25] prefix- and suffix-sequences.

The coding sequence encodes EYFP (enhanced yellow fluorescent protein) which is derived from A. victoria GFP. The excitation is 512 nm and the emission is 534 nm. This part was used to create the parts K1319001 and K1319002. It can also be used in a fusion protein instead of E0030 due to its RFC[25] compability.


K1319001

RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh 1

This part is an RFC [25] dark quencher that is based upon K1319000 (the RFC [25] version of E0030/EYFP). Two mutations were introduced that eliminated fluorescence:

  • L90I
  • Y145W

This protein is designed to be a dark quencher for GFP (E0040) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick K1319013 this is realised and the proteins are fused with the linker K1319016 which includes a specific TEV protease (available as K1319004) cleavage site. The fusion of the proteins bring GFP and REACh 1 in proximity to each other which allows GFP and REACh 1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 1 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows for a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh 1 cancelling the FRET interaction and providing a GFP fluorescence response.


K1319002

RFC [25] - compatible dark quencher based on K1319000 (E0030) called REACh 2

This part is an RFC [25] dark quencher that is based upon K1319000 (the RFC [25] version of E0030/EYFP). Three mutations were introduced that eliminated fluorescence:

  • L90I
  • Y145W
  • H148R

This protein is designed to be a dark quencher for GFP (E0040) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick K1319014 this is realised and the proteins are fused with the linker K1319016 which includes a specific TEV protease (available as K1319004) cleavage site. The fusion of the proteins bring GFP and REACh 2 in proximity to each other which allows GFP and REACh 2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows for a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh 2 cancelling the FRET interaction and providing a GFP fluorescence response.


K1319003

human galectin-3, codon optimized for E. coli

This truncated galectin-3 is a 26 kDa protein that binds certain LPS patterns, present on different pathogens and human erythrocytes. The BioBrick sequence is codon-optimized for E.coli and was submitted in the RFC[25] standard.


K1319004

TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli

This part is a TEV protease in RFC25 that was codon-optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.

The TEV Protease (also known as Tobaco Edge Virus nuclear inclusion a endopeptidase) is a highly sequence specific cysteine protease from the Tobacco Edge Virus (TEV). The protease is highly sequence specific. The consensus sequence for the cut is ENLYFQ\S with \ denoting the cleaved peptide bond. This sequence can be found in the part K1319016.

ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.

The TEV protease is commonly used as a biochemical tool to cleave affinity tags from purified proteins like His-Tags. The high specifity makes the protease relatively non-toxic in vitro and in vivo. The molecular weight of the TEV protease is 27 kDa.


K1319008

IPTG-induced and T7-driven expression of TEV protease

This protein generator produces the TEV protease when induced with IPTG in a DE3 strain or if combined with a T7 RNA-Polymerase generator.


K1319010

Constitutive expression of K1319000

This part expresses K1319000 behind a J23101 constitutive promotor.


K1319011

Constitutive expression of K1319001

This part expresses K1319001 behind a J23101 constitutive promotor.


K1319012

Constitutive expression of K1319002

This part expresses K1319002 behind a J23101 constitutive promotor.


K1319013

Constitutive expression of GFP-REACh1 fusion protein

This part expresses a E0040.K1319001 fusion protein (GFP-REACh1) behind a J23101 promotor. The linker between the two proteins contains a TEV protease cleavage site.


K1319014

Constitutive expression of GFP-REACh2 fusion protein

This part expresses a E0040.K1319002 fusion protein (GFP-REACh1) behind a J23101 promotor. The linker between the two proteins contains a TEV protease cleavage site.


K1319015

Constitutive expression of GFP-EYFP fusion protein

This part expresses a E0040.K1319000 fusion protein (GFP-EYFP) behind a J23101 promotor.


K1319016

TEV protease cleavage site

This sequence codes for a TEV protease cleavage site.

ENLYFQ\S is the optimal cleavage site with the highest activity but the protease is also active to a greater or lesser extent on a range of substrates. The highest cleavage is of sequences closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or medium hydrophobic amino acid and φ is any small hydrophobic amino acid.


K1319017

LasI induced iLOV

This device produces iLOV (K660004) in response to a quorum sensing input.


K1319020

Translational unit of mRFP-galectin3-His

This part is a translational unit of a mRFP-galectin-3-His (B0032.E1010.K1319003.K1319016.B0015).


K1319042

IPTG inducible iLOV

This part can be used for IPTG-induced expression of K660004 (iLOV).


iGEM Team Aachen Biobrick overview

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