Team:Brasil-USP/Parts/KI
BioBrick characterization
Parts
Table of contents
An important part in iGEM competition is to contribute to Registry database with reliable information that hopefully will help other people.
Veg locus promoter: Pveg
Pveg is a strong constitutive promoter well studied in Bacillus subtilis, especially during the vegetative growth phase and sporulation (controlled primarily by the veg gene). Currently available at Registry as BioBrick K823003, it was characterized using a β-galactosidase assay by team LMU-Munich, as well as published in a recent paper [1].
While originally designed for B. Subtilis, during the 2014 iGEM edition Brasil-SP team proposed a protocol to express it in E. Coli (DH5α strain). Repeating this procedure with a gfp gene, we can now perform experiments similar to the β-galactosidase assays and evaluate how strong Pveg is in E. Coli. For instance, we can evaluate it and compare with promoters from the Anderson library [2]. The part and its base BioBricks are represented in figure 1. This question seemed especially interesting given that promoters in the Anderson library were employed in Bacillus subtilis before and Pveg was shown to be 100 times more efficient than J23101 [1], a promoter from the Anderson library we have used as benchmark. We will show that this does not seem to hold for E Coli: Pveg induces less fluorescence that J23101.
Figure 1 - Part used to characterize Pveg constitutive promoter using E. Coli (DH5α strain). Both K823003 and E0840 were parts available in the 2014 iGEM kit. Note that this biobrick was proposed and studied considering Baccilus subtilis originally.
We shall refer to this assembly as KI circuit, using the same nomenclature as Brasil-SP team. In the following sections, we briefly describe our protocols and our main results. Use the Table of Contents in the left panel to quickly navigate.
Results
Discussion
We have shown how Pveg behaves in E. Coli, strain DH5α, and compared it to a benchmark with respect to Anderson Promoter Library: J23101. In fact, this comparison was already been made using Bacillus subtilis and Pveg was demonstrated 100 times stronger than J23101 [1]. However, by changing from Bacillus subtilis chassis to E. Coli’s, we have shown that Pveg loses its overwhelming strength and becomes comparable to J23106 (Device 2 in Interlab Study). This information is potentially useful for any user interested in using Pveg in different chassis, and shows that, when the chassis is changed, a promoter induction may alter substantially.
[1] Radeck et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis, Journal of Biological Engineering, 7:29 2013.
[2] Anderson, J. C. & Team, B. i. Anderson Promoters Colletion,
Protocols - Assembling and Data acquisition