Difference between revisions of "Part:BBa J95022"

 
 
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<partinfo>BBa_J95022 parameters</partinfo>
 
<partinfo>BBa_J95022 parameters</partinfo>
 
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===Characterisation===
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We, Edinburgh_UG 2019 team, decided to characterise this lac based synthetic promoter by assembling it with strong R. sphaeroides RBS (BBa_J95018), GFP (BBa_E0040) and terminator (BBa_B1006) using loop assembly. The promoter and RBS were shown to work in R. sphaeroides with high expression rates (Huo, 2011). We tested if this promoter-RBS combination could yield high expression rates in E. coli as this would provide a convenient system for parallel expression in both R. sphaeroides and E. coli.
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The promoter was found to be strong and leaky, and should be treated as a constitutive promoter unless used in lacIq strains. We observed that induction of E. coli cultures with less than 0.1 OD600 completely inhibited cell growth and resulted in cell death due to metabolic stress. Genetic transformation of R. sphaeroides involves conjugation with E. coli carrying the plasmid of interest.  Leaky expression from such plasmid may cause metabolic stress in E. coli and result in decreased conjugation efficiency.
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[[File:J95023.png|900px|thumb|left|Figure 1. Recombinant E.coli DH5α overnight cultures were diluted to 0.05 OD600 and incubated at 37° to the limit where they had 0.5 OD600. Total of 100μl culture was induced with different IPTG concentrations and measured at an excitation and emission of 485 and 520nm. Orbital averaging was set to 3 and the gain to 1300. Cell growth was measured at 600nm. The fluorescence was divided by the approximated number of cells to get normalised fluorescence per cell.]]

Latest revision as of 12:59, 11 October 2019


PA1/04/03 promoter used for gene expression in R. sphaeroids

promoter for rs241

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]


Characterisation

We, Edinburgh_UG 2019 team, decided to characterise this lac based synthetic promoter by assembling it with strong R. sphaeroides RBS (BBa_J95018), GFP (BBa_E0040) and terminator (BBa_B1006) using loop assembly. The promoter and RBS were shown to work in R. sphaeroides with high expression rates (Huo, 2011). We tested if this promoter-RBS combination could yield high expression rates in E. coli as this would provide a convenient system for parallel expression in both R. sphaeroides and E. coli.

The promoter was found to be strong and leaky, and should be treated as a constitutive promoter unless used in lacIq strains. We observed that induction of E. coli cultures with less than 0.1 OD600 completely inhibited cell growth and resulted in cell death due to metabolic stress. Genetic transformation of R. sphaeroides involves conjugation with E. coli carrying the plasmid of interest. Leaky expression from such plasmid may cause metabolic stress in E. coli and result in decreased conjugation efficiency.

Figure 1. Recombinant E.coli DH5α overnight cultures were diluted to 0.05 OD600 and incubated at 37° to the limit where they had 0.5 OD600. Total of 100μl culture was induced with different IPTG concentrations and measured at an excitation and emission of 485 and 520nm. Orbital averaging was set to 3 and the gain to 1300. Cell growth was measured at 600nm. The fluorescence was divided by the approximated number of cells to get normalised fluorescence per cell.