Team:Cambridge/Project/CA02

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[https://2009.igem.org/Team:Cambridge/Project/Carotenoids/Int Introduction]
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[https://2009.igem.org/Team:Cambridge/Project/CA01 Background]
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[https://2009.igem.org/Team:Cambridge/Project/Carotenoids/PR Previous Research]
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[https://2009.igem.org/Team:Cambridge/Project/CA02 Design]
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[https://2009.igem.org/Team:Cambridge/Project/Carotenoids/OAP Our Action Plan]
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[https://2009.igem.org/Team:Cambridge/Project/CA03 Characterisation]
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[https://2009.igem.org/Team:Cambridge/Project/Carotenoids/RA Result Analysis]
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[https://2009.igem.org/Team:Cambridge/Project/CA05 Datasheet]
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[https://2009.igem.org/Team:Cambridge/Project/Carotenoids/Ref Reference]
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[https://2009.igem.org/Team:Cambridge/Project/CA04 Reference]
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== DESIGN OF DEVICES ==
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== Design of Devices ==
=== Biobricks contributed by previous iGEM teams ===
=== 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:
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:
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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).
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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).
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* 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.
* 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.
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=== Create our own devices by standard assembly ===
=== Create our own devices by standard assembly ===
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* Put the above two constructs under constitutive promoter (R0011) and arabinose-induced promoter/Pbad (I0500) and test the effect on colour production.
* Put the above two constructs under constitutive promoter (R0011) and arabinose-induced promoter/Pbad (I0500) and test the effect on colour production.
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'''''Scheme for standard assembly'''''
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{| style="color:#CCC; background-color:#3D5089;" cellpadding="5" cellspacing="0" border="1"
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!
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! Lycopene-producing device
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! β-carotene-producing device
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|Constituent Biobricks
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(already in the registry)
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|[[Image:Cam09 crtE g.jpg]](K118014)
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 +
[[Image:Cam09 crtB g.jpg]](K118006)
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[[Image:Cam09 crtI g.jpg]](K118005)
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|[[Image:Cam09 crtE g.jpg]](K118014)
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[[Image:Cam09 crtB g.jpg]](K118006)
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[[Image:Cam09 crtI g.jpg]](K118005)
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[[Image:Cam09 crtY g.jpg]](K118013)
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''Basic construct'''
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|[[Image:Cam09 EBI.png|200px]]
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'''K274100'''
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|[[Image:Cam09 EBIY.png]]
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'''K274200'''
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''Under constitutive promoter'''
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|[[Image:Cam09 REBI.png]]
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'''K274110'''
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|[[Image:Cam09 REBIY.png|250px]]
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'''K274210'''
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''Under Pbad promoter'''
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|[[Image:Cam09 IEBI.png|250px]]
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'''K274120'''
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|[[Image:Cam09 IEBIY.png|250px]]
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'''K274220'''
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|}
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 +
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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:
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{| style="color:#CCC; background-color:#3D5089;" cellpadding="5" cellspacing="0" border="1"
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! Biobrick Number
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! Size of insert (kb)
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! Size of backbone (kb)
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! Note
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274100'''
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|3.37
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|2.08
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|Digestion result not shown.
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274110'''
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|3.42
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|2.08
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|
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274120'''
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|4.58
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|4.43
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|Sizes of insert and backbone were similar so only one band appeared on gel.
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274200'''
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|4.53
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|2.08
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|Digestion result not shown.
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274210'''
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|4.59
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|2.08
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|
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''K274210'''
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|5.74
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|4.43
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|
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''R0011-K152005*'''
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|5.48
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|2.08
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|*Used in preliminary research only.
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|'''I0500-K152005*'''
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|6.62
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|4.43
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|*Used in preliminary research only.
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|- style="color:#333; background-color:#FFFFFF;" cellpadding="5" cellspacing="0" border="1"
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|}
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[[Image:Cam09 gel.jpg]]
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Latest revision as of 22:45, 21 October 2009


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 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)

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


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:

Biobrick Number Size of insert (kb) Size of backbone (kb) Note
K274100 3.37 2.08 Digestion result not shown.
K274110 3.42 2.08
K274120 4.58 4.43 Sizes of insert and backbone were similar so only one band appeared on gel.
K274200 4.53 2.08 Digestion result not shown.
K274210 4.59 2.08
K274210 5.74 4.43
R0011-K152005* 5.48 2.08 *Used in preliminary research only.
I0500-K152005* 6.62 4.43 *Used in preliminary research only.

Cam09 gel.jpg

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