Difference between revisions of "Part:BBa K2253000"
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| − | <p> Top10 electrocompetent <i>E. coli</i> cells were transformed with the P8 cassette assembly and plated on LB + CAM plates. After one day of incubation at 37°C, there was growth of purple-blue colonies, which fluoresced red when placed under UV illumination. The observed red fluorescence phenotype was due to the P8 promoter successfully directing expression of the E2-Crimson reporter gene. From this result, we concluded that the P8 constitutive promoter is indeed functional within <i>E. coli</i> and can thus be used to overexpress genes of interest in <i>E. coli</i> as well as <i>L. lactis<i/>. | + | <p> Top10 electrocompetent <i>E. coli</i> cells were transformed with the P8 cassette assembly and plated on LB + CAM plates. After one day of incubation at 37°C, there was growth of purple-blue colonies, which fluoresced red when placed under UV illumination. The observed red fluorescence phenotype was due to the P8 promoter successfully directing expression of the E2-Crimson reporter gene. From this result, we concluded that the P8 constitutive promoter is indeed functional within <i>E. coli</i> and can thus be used to overexpress genes of interest in <i>E. coli</i> as well as <i>L. lactis<i/>. |
Revision as of 02:44, 25 October 2017
Constitutive P8 promoter and RBS composite
The P8 constitutive promoter is natively found Lactococcus lactis. Although the transcriptional efficiency of this promoter has been characterized and tested in Lactococcus lactis and other Gram-positive bacteria, its functionality in Gram-negative species such as E. coli has not been recorded in the literature. We have confirmed that E. coli transformed with a cassette plasmid containing the P8 promoter and E2-Crimson reporter is able to successfully express the E2-Crimson red fluorescent protein. The P32 promoter and RBS sequence are natively found in the genome of Lactococcus lactis. However, we ordered the promoter and RBS sequence as a gBlock using the sequence information provided by Zhu et al. (2015).
Usage and Biology
As a PhytoBrick compatible part, P8 promoter can be assembled into a transcriptional unit via Golden Gate assembly method to upregulate expression of a gene of interest in E. coli as well as L. lactis. of a gene of interest in E. coli as well as L. lactis.
Experimental Design
To test if our Lactococcus lactis constitutive promoters function well within E. coli, we created a test cassette plasmid containing the E2-Crimson reporter gene (which encodes a red fluorescent protein) inserted downstream of the P8 promoter using BsaI Golden Gate assembly. To create this test cassette, we used the P8/P32 promoter part plasmids, E2-Crimson part plasmid, an M13 terminator part plasmid, connector part plasmids, and the pYTK095 vector as the backbone.
Figure 1. Golden Gate assembly process of the P8 test cassette plasmid.
Top10 electrocompetent E. coli cells were transformed with the P8 cassette assembly and plated on LB + CAM plates. After one day of incubation at 37°C, there was growth of purple-blue colonies, which fluoresced red when placed under UV illumination. The observed red fluorescence phenotype was due to the P8 promoter successfully directing expression of the E2-Crimson reporter gene. From this result, we concluded that the P8 constitutive promoter is indeed functional within E. coli and can thus be used to overexpress genes of interest in E. coli as well as L. lactis<i/>.
<p>Figure 2. P8 cassette plasmid transformation plate under normal light (left) and UV illumination (right). </p>
Sequence and Features
References