Difference between revisions of "Part:BBa K3697000"

Latest revision as of 20:40, 27 October 2020

mCherry BSU

This is the coding sequence for producing a codon-optimized mCherry in B. subtilis. mCherry is a derivative of RFP. mCherry_BSU was created by codon-optimizing an E. coli mCherry for expression B. subtilis. When paired with a strong RBS and constitutive promotor (BBa_K3697010), mCherry_BSU can be quantified using a plate reader, and transformed B. subtilis colonies can be moderately red to the naked eye.

mCherry_BSU has an excitation peak at 585 nm and a peak emission at 615 nm. Color is readily visible in E. coli and B. subtilis in under 12 hours of incubation.

Fluorescence levels in B. subtilis and E. coli can be quantified using a fluorescent plate reader. Under high enough expression from a strong constitutive promotor such as pVeg (BBa_K143012), red colonies may be visible to the naked eye. E. coli expressing mCherry_BSU are red under natural light. E. coli liquid cultures may appear red to the naked eye, while B. subtilis liquid cultures likely will not appear red.

This protein can be used as a reporter in B. subtilis due to it's high visibility.

This part does not have a degradation tag.

Usage and Biology

mCherry is a valuable tool for usage in B. subtilis due to its strong potential as a reporter protein. Colonies expression mCherry_BSU at high levels from strong constitutive promotors such as pVeg can appear red to the naked eye. mCherry_BSU can be expressed by both E. coli and B. subtilis, and is not tagged for degredation, giving a strong, enduring signal.

This mCherry was codon optimized for B. subtilis expression. Previous characterization for the Bacillus BioBricks Box 2.0 has shown that wild type B. subtilis strains have very low autofluorescence at wavelengths required to excite mCherry_BSU. With codon optimization, mCherry_BSU has a fluorescent output that is more than 200-fold above autofluorescence. [1]

Due to the high levels of mCherry expression from pVeg, this vector can be used to signal transformation, transcription, and translation in B. subtilis.

Figure 1: E. Coli expressing YFP and mCherry (optimized for B. subtilis). Note: mCherry is being expressed in the E. Coli on the right side of image.

Figure 2: Lawn growth B. subtilis expressing mCherry_BSU

[1] Popp, P.F., Dotzler, M., Radeck, J. et al. The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis . Sci Rep 7, 15058 (2017). https://doi.org/10.1038/s41598-017-15107-z

Sequence and Features

mCherry_BSU Validation in B. subtilis

Stanford iGEM 2020 used this part to test visibility and transformation efficiency of B. subtilis strains 1A976 (xylose-inducible competence) and 1A1276 (mannitol-inducible competence) from the Bacillus Genomic Stock Center.

To test visibility, E. coli transformed with mCherry_BSU were cultured overnight in LB nutrient broth at 37°C. Cells were collected by centrifugation at 3500rpm and mini-prepped. The plasmid DNA from the mini-prep was then transformed into B. subtilis strains 1A976 and 1A1276. Transformed B. subtilis were then added to a 96 well plate at an OD of 1. Fluorescence was measured relative to a blank and a non-transformed control. Liquid cultures were not visibly red to the naked eye.

When plated on kanamycin selective media (20mg/mL), B. subtilis transformed with this vector were mildly red under natural light in less than 24 hours after plating.