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Revision as of 22:44, 17 October 2014

iGEM Team Aachen BioBricks

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

[http://parts.igem.org/Part:BBa_K1319000 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.

[http://parts.igem.org/Part:BBa_K1319001 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 ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319013 K1319013] this is realised and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 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.

[http://parts.igem.org/Part:BBa_K1319002 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 ([http://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [http://parts.igem.org/Part:BBa_K1319014 K1319014] this is realised and the proteins are fused with the linker [http://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [http://parts.igem.org/Part:BBa_K1319004 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.

[http://parts.igem.org/Part:BBa_K1319003 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.

Galectins are proteins of the lectin family, which posess carbonhydrate recognition domains binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3.

Galectin-3 has a size of about 31 kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as cell adhesion, cell growth and differentiation, and contributes to the development of cancer, inflammation, fibrosis and others.

Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including P. aeruginosa protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.

[http://parts.igem.org/Part:BBa_K1319004 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 [http://parts.igem.org/Part:BBa_K1319016 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 [http://parts.igem.org/Part:BBa_K1319007 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.

[http://parts.igem.org/Part:BBa_K1319008 K1319008]

IPTG-induced and T7-driven expression of TEV protease

This protein generator produces the [http://parts.igem.org/Part:BBa_K1319004 TEV protease] when induced with IPTG in a DE3 strain or if combined with a T7 RNA-Polymerase generator.

This biobrick is used in our REACh construct.

[http://parts.igem.org/Part:BBa_K1319010 K1319010]

Constitutive expression of K1319000

This part expresses K1319000 behind a J23101 constitutive promotor.

[http://parts.igem.org/Part:BBa_K1319011 K1319011]

Constitutive expression of K1319001

This part expresses K1319001 behind a J23101 constitutive promotor.

[http://parts.igem.org/Part:BBa_K1319012 K1319012]

Constitutive expression of K1319002

This part expresses K1319002 behind a J23101 constitutive promotor.

[http://parts.igem.org/Part:BBa_K1319013 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 [http://parts.igem.org/Part:BBa_K1319004 TEV protease] [http://parts.igem.org/Part:BBa_K1319016 cleavage site].

[http://parts.igem.org/Part:BBa_K1319014 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 [http://parts.igem.org/Part:BBa_K1319004 TEV protease] [http://parts.igem.org/Part:BBa_K1319016 cleavage site].

[http://parts.igem.org/Part:BBa_K1319015 K1319015]

Constitutive expression of GFP-EYFP fusion protein

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

[http://parts.igem.org/Part:BBa_K1319016 K1319016]

TEV protease cleavage site

This sequence codes for a [http://parts.igem.org/Part:BBa_K1319004 TEV protease] cleavage site.