Difference between revisions of "Part:BBa K1548002"
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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. | ||
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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. | 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. | ||
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Revision as of 03:12, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 202
Illegal AgeI site found at 941
Illegal AgeI site found at 1053 - 1000COMPATIBLE WITH RFC[1000]