Talk:Part:BBa K343001

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Mangler

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- Usage and parameters sektion

Beta-carotene dioxygenase

Retinal synthesis pathway

β,β-carotene-15,15′-dioxygenase is an enzyme that cleaves beta-carotene into two retinal, via the following reaction: Beta-carotene + O(2) <=> 2 retinal (1).

Beta-carotene dioxygenase plays an important role in animal vision, as retinal forms the chemical basis for vision in animals (2). Animals cannot synthesize retinal de novo and thus relies on beta-carotene dioxygenase to transform carotenoids (beta-carotene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin) into retinal (3).

Here we present a biobrick containing this enzyme, and show that it produces retinal both when beta-caroton is added directly to a bacteria containing this BioBrick; and in bacteria double transformated with this BioBrick and the Cambridge 2009 BBa_K274210 (CrtEBIY under constitutive promoter) BioBrick (4) which produces beta-caroten in vivo.

Background

Only two mechanisms for collecting light energy and converting it into chemical energy have been found in nature so fare. The first mechanism is dependent upon photochemical reaction centers (a multisubunit protein complex containing chlorophylls or bacteriochlorophylls, in which light energy is transduced into redox chemistry). The second mechanism uses rhodopsins, retinal-binding proteins that respond to light stimuli (5).

The later mechanism is found extensively througout nature, which has been speculated to be because of the ease, with which this system works; lateral transfer of rhodopsin-based photosystems requires only the genes encoding the rhodopsin apoprotein and a carotenoid oxygenase that produces retinal (5).

The use of rhodopsin photosystes could be of great use in synthetic biology, but this also requires a retinal-forming brick, which we present here.

Retinal

Retinal

Retinal (also called retinaldehyde, vitamin A aldehyde or RAL) has the molecular formular C20H28O and weighs 284.43572 [g/mol]. C 84.45%, H 9.92%, O 5.63%. Melting point at 61-64 degrees celcius. The molecule can exist in four stereoisomeric forms (6).

The apperence of the molecule is orange crystals (in petr ether). UV max is 373 nm (in cyclohexane). Soluble in ethanol, chloroform, cyclohexane, petr ether and oils (6).

Beta-carotene dioxygenase gene from Drosophila melanogaster

The beta-carotene dioxygenase gene (also called "neither inactivation nor afterpotential B" or NinaB for short) was cloned from Drosophila melanogaster cDNA (7). This gene was the first beta-carotene dioxygenase to be molecularly identified and has been intensively studied (8).

The enzyme requires Fe(II) and bile salts as co-factors; in experiments addition of FeSO4 and ascorbat was shown to encrease the activity of the enzyme. In the presence of FeSO4/ascorbate using varying amounts of substrate of beta-caroten (800 pmol to 100 pmol), the apparent Km value of the enzyme was estimated to be 5 mM (8). We speculate that our stains of E. coli contain this cofactors.

The reaction proceeds in three stages, epoxidation of the 15,15′-double bond, hydration of the double bond leading to ring opening, and oxidative cleavage of the diol formed (9).

The protein has a molecular weight of 69.94 kilodaltons.

Usage and parameters

Compatibility

This brick has been tested in the following plasmids and stains:

Chassis: E. coli TOP10, E. coli MG1655.

Plasmids: PSB1C3 (high-copy), PSB3C5 (low-copy).

Devices: Device has been shown to work with BBa_K274210.

Characterization sektion

Hvor hurtigt dannes retinal?


Enzym kinetik med michealis-mentel?

Hvor meget bliver der dannet? (HPLC måling?)

Stabilitet:

Growth rate: OD måling


Risk-assesment

References

  1. ENZYME entry 1.14.99.36 [Internet]. [cited 2010 Oct 13];Available from: http://www.expasy.org/cgi-bin/nicezyme.pl?1.14.99.36
  2. von Lintig J, Dreher A, Kiefer C, Wernet MF, Vogt K. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2001 Jan 30;98(3):1130 -1135.
  3. Retinal - Wikipedia, the free encyclopedia [Internet]. [cited 2010 Oct 13];Available from: http://en.wikipedia.org/wiki/Retinal
  4. Part:BBa K274210 - parts.igem.org [Internet]. [cited 2010 Oct 13];Available from: https://parts.igem.org/Part:BBa_K274210
  5. Bryant DA, Frigaard N. Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol. 2006 Nov;14(11):488-496.
  6. Retinaldehyde - PubChem Public Chemical Database [Internet]. [cited 2010 Oct 13];Available from: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1070
  7. ninaB neither inactivation nor afterpotential B [Drosophila melanogaster] - Gene result [Internet]. [cited 2010 Oct 13];Available from: http://www.ncbi.nlm.nih.gov/gene/41678
  8. von Lintig J, Vogt K. Filling the Gap in Vitamin A Research. Journal of Biological Chemistry. 2000 Apr 21;275(16):11915 -11920.