Part:BBa_K1548002

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. 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. 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.

Drives constitutive expression of mRFP1-6xHisTag protein using the Hsp60 promoter from M. bovis, intended for use in P. acnes (Combination of K1548000 and K1491009).

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]