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=Beta-carotene monooxygenase=

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

Beta-carotene monooxygenase 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 monooxygenase 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 [https://parts.igem.org/Part:BBa_K274210 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===

[[Image:SDU-Denmark-2010-all-trans-retinal.png|130px|thumb|right|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 monooxygenase gene from ''Drosophila melanogaster''===

[[Image:SDU-Denmark-2010-retinal-synthesis.gif‎|200px|thumb|right|Retinal synthesis pathway]]

The beta-carotene monooxygenase 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 monooxygenase to be molecularly identified and studied for it function (8). Formerly this enzyme has been called beta-caroten dioxygenase, but it is now known that only one atom of the dioxygen is incorporated into retinal, hence the name change (9).

The enzyme requires Fe(II) and bile salts as co-factors. In the presence of FeSO4/ascorbate and 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).

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

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).

====Protein structure====

The protein has a calculated molecular weight of 69.94 kilodaltons. The protein structure of beta-caroten monooxygenase is not known. Here we used [http://www.sbg.bio.ic.ac.uk/phyre/html/index.html PHYRE], a protein structure prediction tool, to predict the structure of the enzyme (10):  

<html><head>  
<script language="JavaScript"></script>
<script src=" http://www.sbg.bio.ic.ac.uk/~phyre/qphyre_scripts/Jmol-11.0.1/Jmol.js"></script>  </head>  <body>
 <script> jmolInitialize(" http://www.sbg.bio.ic.ac.uk/~phyre/qphyre_scripts/Jmol-11.0.1/");
jmolApplet(300, "load http://www.sbg.bio.ic.ac.uk/phyre/qphyre_output/8b7ed8b7ea832511/c2biwC_.model.pdb; cpk off; wireframe off;cartoon on; color cartoon group"); 
</script>
</body></html>

==Usage and parameters==

===Usage===

This brick needs beta-caroten to function. In experiments addition of FeSO4 and ascorbat was shown to increase the activity of the enzyme (8). We speculate that normal stains of ''E. coli'' contain these cofactors.


===Performance===

'''Response time''': MANGLER. HVOR HURTIGT OMDANNES BETA-CAROTEN TIL RETINAL? Link til forsøg

'''Production rate''': MANGLER. HVOR MEGET RETINAL DANNES pr. tidsenhed? HPLC FORSØG? EVt. forsøg med forskellige koncentrationer. Link til forsøg

'''Plasmid stability''': MANGLER. Link til forsøgs protokol

'''Growth rate''': MANGLER. OD MÅLING? PFU? Link til forsøgs protokol

'''Enzym kinetics''': KAN IKKE MÅLES PGA HVAD? 

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

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

'''Plasmids''': [https://parts.igem.org/Part:pSB1C3 PSB1C3] (high-copy), [https://parts.igem.org/Part:pSB3C5 PSB3C5] (low-copy).

'''Devices''': Device has been shown to work with [https://parts.igem.org/Part:BBa_K274210 BBa_K274210].

===Safety===
'''General use''': It is our general consensus that this BioBrick does not pose any treat to trained peopled working in a level 1 lab. No special care is needed when working with the BioBrick.

'''Potential pathogenicity''': We do not recommend using this BioBrick for any type of system in humans or animals.

'''Environmental impact''': This BioBrick can be used under controlled settings, but not recommended in the wild. 

Please see our risk assessment as to why we came to these conclusions.

==Risk-assessment==

This risk assessment follows the [SDU-Denmark 2010 Risk Assessment Protocol]. A general risk assessment of our used ''E. coli'' stains can be found at our team [homepage]. 

===General use===
This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people. No special care is needed when working with this BioBrick. 

===Potential pathogenicity===
We do not recommend using this BioBrick for any type of system in humans or animals for the following reasons:
#Retinoic acid, which retinal can degrade into, can affect gene expression and function of almost any cell, including cells of the immune system; it also plays a fundamental role in cellular functions by activating nuclear receptors (11).
#Vitamin A toxicity can lead to hepatic congestion and fibrosis (12).
#Vitamin A and its derivatives have been implicated as chemopreventive and differentiating agents in a variety of cancers (13).

These effects have been observed in humans. Please see references for more information.

===Environmental impact===
Beta-caroten monooxygenase is found in a wide variety of different bacteria, insects and animals (5). As such we would be cautious as to letting a system containing this BioBrick into the wild, since it's function might conflict with existing systems. On the other hand one might argue that since it's function is already available in nature, the function is widely available. 

The product of the BioBrick, retinal, also plays an important function in nature and animals. For this reason we fell that the BioBrick could be used in controlled settings, but not in the wild.

===Construct notes===
1. ''What is the origin of the genetic material used? What does the the genetic materiale do in this origin? Are there uncertainty about the genetical materials function?''

The gene was cloned from ''Drosophila melanogaster'' cDNA. The normal function of the gene is to create beta-caroten monooxygenase as outlined above. The function of this gene is well characterized in the literature and there are little reason to suspect it should function otherwise.


2. ''What modification were done on the genetic materiale before insertion? If anything was modified, what function do you hope to achieve?''

No changes were made to the DNA before inserting it into ''E. coli''.


3. ''What vector did you use? Which antibiotic resistance were involved? Which protocol was used to insert the vector?''

The gene was inserted into two plasmid backbones, both containing chloramphenicol resistance. Both plasmids are specially made for BioBrick use and as such tested and safe. The plasmid was introduced into ''E. coli'' via chemical transformation.


4. ''What is the stability of the vector with respect to genetic traits?''

We have not yet tested the stability of the organism after insertion of our BioBrick.


5. ''How easily can the vector transfer to other bacteria or lifeforms?''

We have not tested the vectors ability to transfer the BioBrick to other bacteria.


6. ''Could other alternatives have been used? Where the safer alternatives?''

We considered the gene, the strains of ''E. coli'' and used plasmids as safe. Cell-free systems might have been used, but these have yet to gain the same function as real bacteria.

==Resources== 

Datasheet for BioBrick.

PDB file for protein structure.


==References==

#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
#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. 
#Retinal - Wikipedia, the free encyclopedia [Internet].  [cited 2010 Oct 13];Available from: http://en.wikipedia.org/wiki/Retinal
#Part:BBa K274210 - parts.igem.org [Internet].  [cited 2010 Oct 13];Available from: https://parts.igem.org/Part:BBa_K274210
#Bryant DA, Frigaard N. Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol. 2006 Nov;14(11):488-496. 
#Retinaldehyde - PubChem Public Chemical Database [Internet].  [cited 2010 Oct 13];Available from: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1070
#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
#von Lintig J, Vogt K. Filling the Gap in Vitamin A Research. Journal of Biological Chemistry. 2000 Apr 21;275(16):11915 -11920.
#ENZYME: 1.14.99.36 [Internet].  [cited 2010 Oct 13];Available from:http://www.genome.jp/dbget-bin/www_bget?ec:1.14.99.36
#Kelley LA & Sternberg MJE. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols. 4, 363 - 371 (2009).
#Spiegl N, Didichenko S, McCaffery P, Langen H, Dahinden CA. Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid. Blood. 2008 Nov 1;112(9):3762-71.
#Russell RM. The vitamin A spectrum: from deficiency to toxicity. American Journal of Clinical Nutrition, Vol. 71, No. 4, 878-884, April 2000.
#Pasquali D, Thaller C, Eichele G. Abnormal level of retinoic acid in prostate cancer tissues. J Clin Endocrinol Metab. 1996 Jun;81(6):2186-91.