Team:Cambridge/Project/CA02

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Carotenoids

DESIGN OF DEVICES

Biobricks contributed by previous iGEM teams

A few iGEM teams of previous years (e.g. Edinburgh 07, Edinburgh 08, Guelph 08) conducted research in the carotenoid system. We are fortunate to have access to some of their Biobricks in the registry:

Registry Code Team Sequence Description Size of insert
K118014 Edinburgh 08 Icon translational unit.png (rbs + CrtE) 922bp
K118006 Edinburgh 08 Icon translational unit.png (rbs + CrtB) 949bp
K118005 Edinburgh 08 Icon translational unit.png (rbs + CrtI) 1498bp. 2 PstI sites removed.
K118013 Edinburgh 08 Icon translational unit.png (rbs + CrtY) 1162bp
K152005 Guelph 08 Icon translational unit.pngIcon translational unit.pngIcon translational unit.pngIcon translational unit.pngIcon reporter.png

(rbs+CrtE) + (rbs+CrtB) + (rbs+CrtI) + (rbs+CrtY) + (rbs+GFP)

5412bp.2 PstI sites in CrtI removed.


The above enzymes all come from a type of Gram negative Enterobacteria, Pantoea ananatis (GenBank: D90087.2). It is noted that analogues of these enzymes are also found in other organisms (e.g. bacteria Rhodobacter spp. and the fungus Neurospora crassa).


During our preliminary research, we encountered a few issues:

  • Originally, the registry annotation for K152005 indicated that there was no ribosome binding site before gene CrtI (i.e. “…rbs+CrtB+CrtI+rbs+CrtY…”). After clarification with Team Guelph 08, we were told that it was an error in annotation. The registry entry for K152005 has since been updated to include the ribosome binding site.
  • Teams of previous years constructed various composite parts containing two or more enzymes of the synthetic pathways (e.g. CrtEBI, CrtEBIY), but they are not available on the 2009 Distribution Plates.
  • We put K152005 under control of constitutive promoter (R0011) and transformed the construct into E.coli TOP10 cells. However, the plasmids seemed unstable (the colonies streaks on solid agar plates were inconsistent in colour) and the pigment production was low. This indicated that a different strain of E.coli might be needed for pigment production.


Create our own devices by standard assembly

Given the information at hand, we decided to adopt the following general strategies:

  • Create a lycopene-producing device (with enzymes CrtE, CrtB and CrtI) by standard assembly of Biobricks K118014, K118006 and K118005.
  • Create a β-carotene-producing device (with enzymes CrtE, CrtB, CrtI and CrtY) by standard assembly of Biobricks K118014, K118006, K118005 and K118013.
  • Put the above two constructs under constitutive promoter (R0011) and arabinose-induced promoter/Pbad (I0500) and test the effect on colour production.
Lycopene-producing device β-carotene-producing device
Constituent Biobricks

(already in the registry)

Cam09 crtE g.jpg(K118014)

Cam09 crtB g.jpg(K118006)

Cam09 crtI g.jpg(K118005)

Cam09 crtE g.jpg(K118014)

Cam09 crtB g.jpg(K118006)

Cam09 crtI g.jpg(K118005)

Cam09 crtY g.jpg(K118013)

Basic construct Cam09 EBI.png K274100 Cam09 EBIY.png K274200
Under constitutive promoter Cam09 REBI.png K274110 Cam09 REBIY.png K274210
Under Pbad promoter Cam09 IEBI.png K274120 Cam09 IEBIY.png K274220


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