Difference between revisions of "Part:BBa K3182300"
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[[File:T--Linkoping_Sweden--mgnen.png|420px|thumb|left|<b>Figure 2.</b>Comparison of the old biobrick, BBa_K2671000 (light grey), and the new biobrick BBa K3182300 (dark grey). Relative fluorescence after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.]]
 
[[File:T--Linkoping_Sweden--mgnen.png|420px|thumb|left|<b>Figure 2.</b>Comparison of the old biobrick, BBa_K2671000 (light grey), and the new biobrick BBa K3182300 (dark grey). Relative fluorescence after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.]]
  
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To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD600) of 0.4.
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To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD600) of 0.4 ± 0.05. Both biobricks were inoculated in LB-Miller media containing 25 ug/mL chloramphenicol. After the cultures reached OD600 0.4, this part was induced using 0.5 mM IPTG and the old part was in induced with 1.5 mM L-arabinose. Aliquotes of the induced cultures were then placed in a 96-well plate and later analyzed with spectrometry. Fluorescence was then measured over 16 hours and excitation wavelength was
  
 
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Revision as of 10:44, 18 October 2019

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 91
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

pT7-mNG

This is a improved variant of BBa_K2671000. The biobrick codes for mNeonGreen (mNG), which is a fluorescent protein with great intensity. The protein is currently ranked third in intensity, only beaten by mVenus-Q69M (basic) and Skylan-S (photoswitchable).

This part has a T7-system as well as a 5’-UTR region, instead of the AraC-pBAD system present in the non-improved biobrick BBa_K2671000. By using the T7-DNA-directed-RNA-polymerase (T7-DdRNAP) from a bacteriophage over the native DNA-directed-RNA-polymerase (n-DdRNAP) in E. coli the expression is greatly increased. This part requires a host carrying the T7-DdRNAP, such as E. coli BL21 (DE3) which was the chassis we used.


Figure 1. A schematic representation of the improvement. Where the promotor (purple, pBAD) of the previous biobrick (BBa_K2671000) has been exchanged to pT7. Translation enhancing '5-UTR containing g10-L RBS (BBa_K1758100) has been added as well to the improvement. Together with removal of extra bases (named UTR in figure 1) which is non-coding. A BamHI site was added after the His-tag. The blue colors illustrate the protein coding part of the biobricks.

























Usage and Biology


Figure 2. A spectral scan of mNeonGreen. Purified mNeonGreen was analyzed with spectrometry to find optimal excitation and emission wavelengths.
Figure 2.Comparison of the old biobrick, BBa_K2671000 (light grey), and the new biobrick BBa K3182300 (dark grey). Relative fluorescence after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.

To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD600) of 0.4 ± 0.05. Both biobricks were inoculated in LB-Miller media containing 25 ug/mL chloramphenicol. After the cultures reached OD600 0.4, this part was induced using 0.5 mM IPTG and the old part was in induced with 1.5 mM L-arabinose. Aliquotes of the induced cultures were then placed in a 96-well plate and later analyzed with spectrometry. Fluorescence was then measured over 16 hours and excitation wavelength was