Difference between revisions of "Part:BBa K1548002"

Latest revision as of 03:13, 18 October 2014

Hsp60 Promoter-RBS-mRFP1-6xHisTag Strong constitutive promoter from M. bovis which has also shown to function in a variety of mycobacteria (1) drives constitutive expression of mRFP1-6xHisTag protein using the Hsp60 promoter from M. bovis (Combination of K1548000 and K1491009). It is expected to function in P. acnes a closely-related propionibacterium. The promoter does not function in E. coli, as we determined by an mRFP1 expression assay in the context of Part:BBa_K1548002, https://parts.igem.org/Part:BBa_K1548002 (Figure 1). This negative result was confirmed by E. coli promoter prediction software BPROM on (http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) which did not detect appropriate -10 and -35 E. coli promoter elements in the hsp60 promoter DNA sequence, validating our negative result.

Figure 1.The hsp60 promoter does not produce mRFP1 in E. coli. E. coli cells containing the hsp60-RBS-mRFP1 plasmid or hsp60-alone negative control plasmid were grown O/N and then diluted 1:250, grown for 4hrs diliuted to equal OD600 and assayed for mRFP1 fluoresecence in a fluorescence plate reader. n=3 and Stdev is plotted. References: 1. Oldfield LM, Hatfull GF. Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression. J Bacteriol. 2014 Oct 15;196(20):3589-97. doi: 10.1128/JB.01801-14. Epub 2014 Aug 4.

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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 202
Illegal AgeI site found at 941
Illegal AgeI site found at 1053
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K1548002 short</partinfo>
-Strong constitutive promoter from M. bovis which has also shown to function in a variety of mycobacteria (1) drives constitutive expression of mRFP1-6xHisTag protein using the Hsp60 promoter from M. bovis (Combination of K1548000 and K1491009). It is expected to function in P. acnes a closely-related propionibacterium. The promoter does not function in E. coli, as we determined by an mRFP1 expression assay in the context of Part:BBa_K1548002, https://parts.igem.org/Part:BBa_K1548002 (Figure 1). This negative result was confirmed by E. coli promoter prediction software BPROM on (http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) which did not detect appropriate -10 and -35 E. coli promoter elements in the hsp60 promoter DNA sequence, validating our negative result.
+Strong constitutive promoter from M. bovis which has also shown to function in a variety of mycobacteria (1) drives constitutive expression of mRFP1-6xHisTag protein using the Hsp60 promoter from M. bovis (Combination of K1548000 and K1491009). It is expected to function in P. acnes a closely-related propionibacterium. The promoter does not function in E. coli, as we determined by an mRFP1 expression assay in the context of Part:BBa_K1548002, https://parts.igem.org/Part:BBa_K1548002 ('''Figure 1'''). This negative result was confirmed by E. coli promoter prediction software BPROM on (http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) which did not detect appropriate -10 and -35 E. coli promoter elements in the hsp60 promoter DNA sequence, validating our negative result.
 [[File:Hsp60.png]]
-Figure 1.The hsp60 promoter does not produce mRFP1 in E. coli. E. coli cells containing the hsp60-RBS-mRFP1 plasmid or hsp60-alone negative control plasmid were grown O/N and then diluted 1:250, grown for 4hrs diliuted to equal OD600 and assayed for mRFP1 fluoresecence in a fluorescence plate reader. n=3 and Stdev is plotted.
+'''Figure 1.The hsp60 promoter does not produce mRFP1 in E. coli.''' E. coli cells containing the hsp60-RBS-mRFP1 plasmid or hsp60-alone negative control plasmid were grown O/N and then diluted 1:250, grown for 4hrs diliuted to equal OD600 and assayed for mRFP1 fluoresecence in a fluorescence plate reader. n=3 and Stdev is plotted.
 References: 1. Oldfield LM, Hatfull GF. Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression. J Bacteriol. 2014 Oct 15;196(20):3589-97. doi: 10.1128/JB.01801-14. Epub 2014 Aug 4.