As a contribution to the parts registry, we decided to do more research on the Lux cassette right promoter [Part: BBa_I1051]. Here, we give more details about what the Lux cassette right promoter is, an approach to using this promoter, a mathematical model provided by a paper, and discoveries related to this part. We believe these are useful pieces of information needed to understand a part and how one might use it themselves. Here is the link to the relevant part page.
Lux cassette is a unique member of the TetR superfamily of transcription factors [1]. Due to their ability to activate and repress large regulons of genes, bacterial Lux system is used as gene expression reporter and regulators for quorum sensing [1]. Benefits of using this system is its speed, sensitivity and non-destructivity [2]. However, due to its nonlinear molecular and enzyme dynamics, Lux cassette’s bioluminescence is different from gene expression [2]. This causes limitations in bioluminescent data interpretation.
According to research done by Mudassar Iqbal in 2017, computational approaches could enable researchers to infer gene transcription levels of Lux cassette right promoters. Previously, bioluminescent profiles resulted in transient peaks of light output being different in experimental data sets compared to gene expression [2]. Therefore, using Bayesian inference schemes, researchers are able to reverse engineer promoter activity from the bioluminescence [2].
Figure 1. Mathematical model for LUX system
Using this mathematical approach, researchers were able infer LUX right promoter activity from light readout. The Lux right promoter input function is modeled as a series of K heights at fixed positions. Using a model proposed by Green in1995, researchers were able to measure the distribution for each height at point n with mean value equal to the current height at n-1. Points are chosen at random and new heights are proposed accordingly [2]. Using the Gaussian error model, a likelihood function is proposed [2]. This approach is crucial as it enables normalization of light values for the Lux system including the Lux right promoter [2].
Data from this research provides evidence that decrease in bioluminescence can be interpreted as a switching off of the promoter, while an increase in bioluminescence would be better interpreted as a longer period of gene expression [2].
The terminator t1 is located approx. 280 nucleotides beyond the int gene of bacteriophage k with role of both transcription terminator as well as provider of stability achieved by protecting int message from 3' exonucleolytic degradation [1]. Studies investigating t1 sequence in transcription termination and RNA stability induced point mutations in order to map G + C-rich region of dyad symmetry in the terminator and decrease its transcriptional termination [1]. Additionally, the tI mutations cause upstream transcript instability in vivo which is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts [1]. These results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase [1].
Lambda tI is an intrinsic terminator, with an interrupted dyad symmetry followed by a run of uridine residues. Percentage thymine of tI is increased when the MgCl2 concentration is decreased in the in vitro reaction which is consistent with other intrinsic terminators [2]. However, λ tI has an unusual structure in that the hairpin helix is almost 50% A-T and G-C base pairs, and the loop contains a sequence of 5 U residues [4]. Despite that they are multiple, only one to two termination sites are seen upon their mapping. This is unusual since part of the stem is mainly A-U and therefore the secondary structure is mainly found within the RNA hairpin stem [2]. Lastly, tI termination sites occur in the uridine residues with some heterogeneity, and they overlap with a retroregulation structure that triggers RNA processing by RNase III and PNPase [2]. Therefore, tI is a complex transcriptional terminator where protein-terminator interactions might be expected [2].
The tI point mutations reduced transcription termination at tI and induced transcript instability of those messenger RNAs that do terminate at tI [1]. The in vivo stability of the tI mRNA is determined by the intact secondary structure of tI and the presence of an active PNPase [1]. Stem Loop structure of tI is necessary for both transcription termination and RNA stability, and point out the important role of tI int expression [1]. The 3’-untranslated trailer region af the int mRNA is unusually long [3].