Difference between revisions of "Part:BBa J153002"

Latest revision as of 14:00, 13 February 2012

Ptrc2O

Ptrc2O is a modified version of Ptrc1O (Part:BBa_J153001) that contains two lac operators to allow for stronger repression through DNA looping by the LacI tetramer.

It contains the lac O1 operator (proximal to the core promoter) and the ideal/symmetrical lac operator (distal to the core promoter) that allow for LacI repression and subsequent induction by IPTG.

Usage and Biology

It was characterized with a GFP reporter construct (Part:BBa_I13504) carried on the broad-host-range shuttle vector pPMQAK1 (Part:BBa_J153000) to be of similar strength and repressibility as Ptrc1O in E. coli DH5alpha. Characterization in the cyanobacterium Synechocystis sp. PCC 6803 revealed strong fluorescence from the Ptrc2O-driven construct, similar to the Ptrc1O-driven construct. However, as compared to Ptrc1O, it was strongly repressed by LacI, but could on the other hand not be induced by IPTG to its full strength (Huang etal 2010) [http://www.ncbi.nlm.nih.gov/pubmed/20236988].

When cloning expression constructs driven by Ptrc2O, or just the promoter itself, in a strain not expressing the lac repressor, promoter mutations were frequently observed. Hence it's a good idea to use this promoter in combination with a LacI-expressing E. coli strain.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Revision as of 14:00, 13 February 2012 (view source) Daniel Camsund (Talk \| contribs) m (→‎Usage and Biology) ← Older edit		Latest revision as of 14:00, 13 February 2012 (view source) Daniel Camsund (Talk \| contribs) m (→‎Usage and Biology)
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	It was characterized with a GFP reporter construct ([[Part:BBa_I13504]]) carried on the broad-host-range shuttle vector pPMQAK1 ([[Part:BBa_J153000]]) to be of similar strength and repressibility as P''trc1O'' in ''E. coli'' DH5alpha. Characterization in the cyanobacterium ''Synechocystis'' sp. PCC 6803 revealed strong fluorescence from the P''trc2O''-driven construct, similar to the P''trc1O''-driven construct. However, as compared to P''trc1O'', it was strongly repressed by LacI, but could on the other hand not be induced by IPTG to its full strength (Huang etal 2010) [http://www.ncbi.nlm.nih.gov/pubmed/20236988].		It was characterized with a GFP reporter construct ([[Part:BBa_I13504]]) carried on the broad-host-range shuttle vector pPMQAK1 ([[Part:BBa_J153000]]) to be of similar strength and repressibility as P''trc1O'' in ''E. coli'' DH5alpha. Characterization in the cyanobacterium ''Synechocystis'' sp. PCC 6803 revealed strong fluorescence from the P''trc2O''-driven construct, similar to the P''trc1O''-driven construct. However, as compared to P''trc1O'', it was strongly repressed by LacI, but could on the other hand not be induced by IPTG to its full strength (Huang etal 2010) [http://www.ncbi.nlm.nih.gov/pubmed/20236988].

−	When cloning expression constructs driven by P''~~trc1O~~'', or just the promoter itself, in a strain not expressing the ''lac'' repressor, promoter mutations were frequently observed. Hence it's a good idea to use this promoter in combination with a LacI-expressing ''E. coli'' strain.	+	When cloning expression constructs driven by P''trc2O'', or just the promoter itself, in a strain not expressing the ''lac'' repressor, promoter mutations were frequently observed. Hence it's a good idea to use this promoter in combination with a LacI-expressing ''E. coli'' strain.