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Overview Sensitivity Tuner --- Characterisation --- Modelling Colour Generators --- Carotenoids (Orange/Red) --- Melanin (Brown) --- Violacein (Purple/Green) The Future Safety

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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	(rbs + CrtE)	922bp
K118006	Edinburgh 08	(rbs + CrtB)	949bp
K118005	Edinburgh 08	(rbs + CrtI)	1498bp. 2 PstI sites removed.
K118013	Edinburgh 08	(rbs + CrtY)	1162bp
K152005	Guelph 08	(rbs+CrtE) + (rbs+CrtB) + (rbs+CrtI) + (rbs+CrtY) + (rbs+GFP)	5412bp.2 PstI sites in CrtI removed.

The above enzymes all come from a 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.

Scheme for standard assembly

	Lycopene-producing device	β-carotene-producing device
Constituent Biobricks (already in the registry)	(K118014) (K118006) (K118005)	(K118014) (K118006) (K118005) (K118013)
Basic construct	K274100	K274200
Under constitutive promoter	K274110	K274210
Under Pbad promoter	K274120	K274220

After constructing our devices, we conducted restriction digest (cut with enzymes XbaI and PstI on Biobrick prefix and suffix, respectively) to check that the sizes of the inserts were correct. The results are as follows: