Difference between revisions of "Part:BBa K3697010"

Latest revision as of 20:41, 27 October 2020

mCherry_BSU Plasmid

This part is an expression vector for Bacillus subtilis that produces codon-optimized mCherry_BSU, for integration into Bacillus subtilis at the AmyE loci (BBa_K143001). mCherry_BSU is under pVeg expression (BBa_K143012), the strongest constitutive promotor known in B. subtilis. Plasmid expresses kanamycin resistance in B. subtilis, and ampicillin resistance for cloning in E. coli.

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.

This plasmid successfully integrates into B. subtilis and produces mCherry_BSU at levels that can be quantified using a fluorescent plate reader. Transformation was performed using xylose inducible competent B. subtilis, and plated on selective kanamycin media (20ug/mL). When cloning in E. coli, the E. coli may be red, as they will non-discriminately express the mCherry_BSU. E. coli transformed with this plasmid have been clearly red under natural light for the duration of their lifetime and does not appear to degrade. Transformed B. subtilis colonies may appear weakly 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.

The RBS for expression of mCherry in this vector is strong.

The mCherry does not have a degredation 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

This part contains an AmpR CDS for cloning in E. coli. mCherry_BSU is under pVeg expression, coupled with a strong RBS and terminator. In the reverse direction, this part contains KanR for selection in B. subtilis. The pVeg, mCherry, and KanR are all contained within AmyE integration sites, allowing for recombination of the plasmid into the subtilis genome.

mCherry_BSU Plasmid 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 the mCherry_BSU plasmid 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 colonies transformed with this vector were mildly red under natural light in less than 24 hours after plating.