Part:BBa_K3059424

Strong Constitutive mScarlet-I expressing circuit in M. smegmatis

Strong Constitutive mScarlet-I expressing circuit in Mycobacterium smegmatis. Components: S16 promoter mScarlet-I

Characterization

Image of Mycobacterium smegmatis transformed with BBa_K3059424 under inverted fluorescence microscope:

Magnification: 1000X. Filter: RGB-TRITC -> Transmission -> RGB

Negative Control with untransformed Mycobacterium smegmatis:

Magnification: 1000X. Filter: RGB-TRITC -> Transmission -> RGB

Function Conparison with BBa_K3059423

Fluorescence intensity normalized with respect to OD600 plotted on a log scale. Each dot represents a distinct biological replicate (colonies). Negative control measures untransformed, nonfluorescent M.smeg mc2155 while blank measures 7H9 media with no inoculation.

As shown in this graph, BBa_K3059424 increases fluorescence intensity by up to 1500X fold comparing to background fluorescence.

Improvement

For our improved part this year, we improved BBa_K2333413 by switching out original promoter, ribosome binding site and terminator for Mycobacterium compatible parts. BBa_K2333413 is a constitutive mScarlet-I expressing circuit, with BBa_J23100 as its promoter. BBa_K3059424 also expresses mScarlet-I, which is driven by a relatively strong Mycobacterium promoter S16 (BBa_K3059410). We achieved similar fluorescence intensity level by transforming BBa_K3059424 into Mycobacterium smegmatis; Normalized fluorescence intensity data is plotted in the following graph:

Fluorescence intensity normalized with respect to OD600 plotted on a log scale. Each dot represents a distinct biological replicate (colonies). Negative control measures untransformed, nonfluorescent M.smeg mc2155.

We can see that BBa_K3059424 showed slightly lower fluorescence intensity measurements than BBa_K2333413, but still highly notable. This circuit could also serve as a framework for engineering mycobacterium using plasmids, since mScarlet-I could easily be switched out to any gene of interest.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 765
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]