Difference between revisions of "Part:BBa K3697009"

Latest revision as of 20:42, 27 October 2020

pVeg YFP Plasmid for B. subtilis

This part is an expression vector for Bacillus subtilis that produces codon-optimized YFP, for integration into Bacillus subtilis at the AmyE loci (BBa_K143001). YFP 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.

YFP has an excitation peak is 514 nm and an emission peak is 527 nm.

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

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

The YFP does not have a degradation tag.

Usage and Biology

YFP is a valuable tool for usage in B. subtilis due to its strong potential as a reporter protein. Colonies expression YFP at high levels from strong constitutive promotors such as pVeg can be quantified using a plate reader. This YFP can be expressed by both E. coli and B. subtilis, and is not tagged for degredation, giving a strong, enduring signal.

Due to the high levels of YFP expression from pVeg, this vector can be used to signal transformation, transcription, and translation in B. subtilis. When this plasmid is transformed into B. subtilis, they should express YFP at levels that can be quantified using a fluorescent plate reader.

This vector integrates into the AmyE gene in the B. subtilis genome, which codes for a nonessential α-amylase.

Stanford iGEM 2020 used this plasmid as a backbone for an RNA toehold based detection system. The plasmid was linearized using PCR such that one end of the plasmid was pVeg and the other end was YFP. The insert, containing an the sequence encoding a toehold (BBa_K3697011), was flanked by 40bp of homology to pVeg, and 40bp homology to YFP. The linearized pVeg YFP backbone and the insert were then combined using Gibson assembly. The resulting product should be a toehold that regulates the translation of YFP, and is under pVeg expression. In the presence of the KanR gene or RNA, the toehold binds, enabling translation of the YFP.

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

Sequence and Features