Designed by: Tobias Hensel Group: iGEM18_Marburg (2018-09-18)
Phytobrick version of Lux Operon
This is the Phytobrick version of the coding sequence Lux and was build as a part of the Marburg Collection. Instructions of how to use the Marburg Collection are provided at the bottom of the page.
Open reading frames (ORFs) are sequences that can theoretically be translated into functional proteins. They are predicted by a start codon (ATG) at their beginning and a stop codon (TAA, TAG, TGA) at the end separated by multiple codons. When an ORF is confirmed to code for a functional protein it is classified as a coding sequence (CDS). CDSs code only proteins. To translate them they need a promoter upstream the start codon.A terminator downstream of the sequence end the translation. In prokaryotes an RBS sequence is needed between the promoter and the start codon. In eukaryotes CDSs are flanked by untranslated regions (UTRs). In addition eukaryotic CDSs contain introns which are removed after transcription by splicing to form the mature mRNA which is translated into the functional protein outside the nucleus.
Figure 1: Mean ratio of reporter signal over medium blank during the coarse of the experiment.
After having established a reliable workflow for V. natriegens, we investigated four different reporters and measured the signal to blank ratio. Test constructs (shown in figure 2) were built by using the same set of parts except for the coding sequence. sfGFP, RFP, YFP and the lux operon were analyzed for their performance in V. natriegens. The best signal to blank ratio by far was achieved for the lux operon (2000), followed by sfGFP (3), RFP (1) and YFP (no detectable signal). The main explanation for the superior performance of the lux operon is the almost complete absence of background signal without reporter expression. This makes the lux operon a perfect reporter that can even be used to analyze extremely low levels of expression caused by very weak promoters or terminator read through. Based on this finding, we decided to use the lux operon as our reporter for all subsequent experiments.