Composite

Part:BBa_K3889131

Designed by: Prameya Garge, Ashwin Sharma   Group: iGEM21_IISER-Tirupati_India   (2021-10-14)


Bacillus subtilis BBa_B0010 terminator check cassette

BBa_B0010 Terminator check cassette for use in Bacillus subtilis. T--IISER-Tirupati India--Improvement2.jpeg
Fig 1. Spacer replaced from BBa_K3889130 by BBa_B0010

Formulae for terminator efficiency [1]:

T--IISER-Tirupati India--Eq1.png

where

mCherry0 → mCherry produced by device without terminator

sfGFP0 → sfGFP produced by device without terminator

Using the device (BBa_K3889130) without any changes, TEDevice can be calculated which gives the expression of mCherry in absence of a terminator.

T--IISER-Tirupati India--Eq2.png

where

mCherry → mCherry produced by the device with terminator

sfGFP → sfGFP produced by the device with terminator

and TE → is the terminator efficiency of the BBa_B0010

Results

T--IISER-Tirupati India--Bsfield1.png

Fig. Field 1: a. B.subtilis 168 with B0010 Terminator Check Cassette-green fluoresence at wavelength 498nm (under magnification 100X) b. B.subtilis 168 with B0010 Terminator Check Cassette in bright field (under magnification 100X) c. B.subtilis 168 with B0010 Terminator Check Cassette-red fluoresence at wavelength 587nm (under magnification 100X)

T--IISER-Tirupati India--Bsfield2.png
Fig. Field 2: a. B.subtilis 168 with B0010 Terminator Check Cassette-green fluoresence at wavelength 498nm (under magnification 100X) b. B.subtilis 168 with B0010 Terminator Check Cassette in bright field(under magnification 100X) c. B.subtilis 168 with B0010 Terminator Check Cassette-red fluoresenceat wavelength 587nm (under magnification 100X) d. B.subtilis 168 with B0010 Terminator Check Cassette at wavelength 383nm (under magnification 100X)

Under a confocal microscope, we could observe the expression of sfGFP and mCherry and observe their differences in fluorescence intensities signifying that BBa_B0010 works in Bacillus subtilis.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 121


References

1. Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011

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