Difference between revisions of "Part:BBa K1820017"
 
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===Usage and Biology===
 
===Usage and Biology===
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*This has only been tested in ''E. coli'' thus far.
  
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<p> This is a composite part designed as a report protein sequence for use in ''Lactococcus lactis''. The promoter is a mid-range promoter from Peter Ruhdal Jensen and Karin Hammer's library of synthetic promoters for ''Lactococcus lactis'' followed by a popular ribosome binding site (Elowitz 1999), an mCherry fluorescent protein codon optimized for ''Lactobacillus reuteri'' and biobricked according to standard 25, and a popular double-stop terminator. Although it was designed for ''L. lactis'', it has displayed function in ''Escherichia coli'' as well. There are indications that the promoter is functional in a large number of prokaryotic organisms. As such, it is likely that this construct will be functional in a variety of prokaryotic organisms.</p>
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<p> This is a composite part designed as a report protein sequence for use in ''Lactococcus lactis''. The promoter is a mid-range promoter, part BBa_K1033220 from the 2013 Uppsala team, from Peter Ruhdal Jensen and Karin Hammer's library of synthetic promoters for ''Lactococcus lactis'' followed by a popular ribosome binding site (Elowitz 1999), an mCherry fluorescent protein, part BBa_K1033250 from the 2013 Uppsala team, codon optimized for ''Lactobacillus reuteri'' and biobricked according to standard 25, and a popular double-stop terminator. We further characterized this promoter with the addition of a fluorescent protein. Although it was designed for ''L. lactis'', it has displayed function in ''Escherichia coli'' as well. There are indications that the promoter is functional in a large number of prokaryotic organisms. As such, it is likely that this construct will be functional in a variety of prokaryotic organisms.</p>
  
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<p>We created and tested this construct in ''Escherichia coli'' in the pSB1C3 plasmid. It was tested for fluorescence relative to non-transformed ''E. coli'' cell and two other similar constructs (see Figure 1).</p>
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<p>We created and tested this construct in ''Escherichia coli'' in the pSB1C3 plasmid. It was tested for fluorescence relative to non-transformed ''E. coli'' cells (see Figure 1).</p>
  
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<p>This part improves upon CP8 (https://parts.igem.org/Part:BBa_K1033220) by the addition of a RBS, fluorescent protein, and a terminator, as well as further characterization of the promoter.</p>
  
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https://static.igem.org/mediawiki/parts/1/1b/Utah_State_2015_MCherry_Fluorescence_Chart.jpg
 
  
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<p><strong> Figure 1. </strong> Fluorescence levels from three constructs using synthetic promoters from Jensen and Hammer (1998) in ''E. coli'' excited at 530/25 with emissions read at 590/35. cp8 from part BBa_K1820017, cp11 from part BBa_K1820018, and cp44 from part BBa_K1820019</p>
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https://static.igem.org/mediawiki/2015/e/e1/Utah_State_2015_CP8_mCherry%28Lr%29.jpeg
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<p><strong> Figure 1. </strong> Fluorescence levels from BBa_K1820017 excited at 530/25 with emissions read at 590/35</p>
  
 
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Latest revision as of 23:27, 27 September 2015

CP8_RBS_mCherry(Lr)_Terminator

Usage and Biology

  • This has only been tested in E. coli thus far.

This is a composite part designed as a report protein sequence for use in Lactococcus lactis. The promoter is a mid-range promoter, part BBa_K1033220 from the 2013 Uppsala team, from Peter Ruhdal Jensen and Karin Hammer's library of synthetic promoters for Lactococcus lactis followed by a popular ribosome binding site (Elowitz 1999), an mCherry fluorescent protein, part BBa_K1033250 from the 2013 Uppsala team, codon optimized for Lactobacillus reuteri and biobricked according to standard 25, and a popular double-stop terminator. We further characterized this promoter with the addition of a fluorescent protein. Although it was designed for L. lactis, it has displayed function in Escherichia coli as well. There are indications that the promoter is functional in a large number of prokaryotic organisms. As such, it is likely that this construct will be functional in a variety of prokaryotic organisms.

We created and tested this construct in Escherichia coli in the pSB1C3 plasmid. It was tested for fluorescence relative to non-transformed E. coli cells (see Figure 1).

This part improves upon CP8 (https://parts.igem.org/Part:BBa_K1033220) by the addition of a RBS, fluorescent protein, and a terminator, as well as further characterization of the promoter.


Utah_State_2015_CP8_mCherry%28Lr%29.jpeg

Figure 1. Fluorescence levels from BBa_K1820017 excited at 530/25 with emissions read at 590/35


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]


References

Jensen, P. R., Hammer, K. (1998). The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl Environ Microbiol. 1998 Jan; 64(1): 82–87. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/

mCherry Fluorescent Protein. Clonetek, Takara Bio. http://www.clontech.com/US/Products/Fluorescent_Proteins_and_Reporters/Fluorescent_Proteins_by_Name/mCherry_Fluorescent_Protein. Accessed 17 Sept 2015.