Difference between revisions of "Part:BBa K4140005"

Latest revision as of 18:50, 11 October 2022

L7AE

Part Description

A protein called L7Ae that binds to RNA inhibits the targeted transcript's ability to be translated. Transcriptional repressors take longer to suppress the intended construct than L7Ae which regulates gene expression at the translational level. As it specifically targets a sequence on the 5' end of the RNA termed the 2x-k-turn. L7Ae can be used to build more complicated genetic circuits that are regulated at both the translational and transcriptional levels

Usage

As a post-transcriptional modifier, we employed it to repress the translation of cas12g protein in case of high levels of phenylalanine and TyrR that leads to repressing CRISPR system activity and preserving the expression of PAH in the therapeutic circuit or lacZ alpha in the diagnostic circuit as shown in figure 1.

Figure 1. (shows the usage of TL7Ae in our circuit design)

Characterization of Mutational Landscape

After creating a multiple sequence alignment of the protein sequence and predicting mutational landscapes, the effect of these mutations on the evolutionary fitness of the protein is tested. The prediction of the mutational landscape by saturation mutagenesis of the L7Ae protein. The (N99D) mutation, as depicted in the chart, had the greatest score when compared to other mutations. On the other hand, it's clear that the (A60M) had the least evolutionary fitness for LacZ protein. As displayed in Figure(2)

Figure 2. (shows the mutational landscape of the L7Ae protein)

Literature Characterization

In order for our regulatory CRISPR-based system, which L7Ae attaches to, to operate as a regulator, domain the RNA of k-turn should be unstructured in the absence of L7Ae. In this figure, we examine how L7Ae affects both the native sequence and the mutant version of the k-turn. and in-line probing for both original and mutant RNA at concentrations of 0, 2.5, 5, and 10 M AfL7Ae, respectively. Positions of reactivity in the native 5′-UTR RNA in the presence of L7Ae are shown by the black circles drawn on the fluorogram as shown in figure 3

Figure.3 Using in-line probing, analyze the conformational change in the A. fulgidus 5′-UTR RNA caused by the interaction of AfL7Ae protein.

Characterization by mathematical modeling

This model is to simulate the kinetics of the riboswitch (L7Ae with kink turns) that is used in our circuit. The designed circuit is to detect if the increasing substance is either phenylalanine or tyrosine via TyrR. So if phenylalanine level is elevated, L7Ae is formed as it is downstream TyrR that forms a complex via binding with its kink-turn on another circuit; that complex inhibits expression of cas12g, so the circuit will be able to express lacZ alpha (beta-galactosidase) in diagnostic circuit or PAH in the therapeutic circuit. If tyrosine level is elevated with a decreased level of phenylalanine, it activates tyrR inhibitory promoter so no L7Ae would be expressed resulting in cas12g expression to control the circuit as shown figure (4) and graph (1).

Figure (4) illustrates the kinetics of all reactions in riboswitch model

Graph (1) illustrates riboswitch kinetics in which Q represents the condition where L7Ae is expressed and bound to its kink-turns ,therefore inhibiting the expression of cas12g. However, M represents no expression of L7Ae in which cas12g would be expressed to control the circuit if the phenylalanine is absent.

Experimental Characterization

This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 5. The runnning part (ordered from IDT) included ParoF promoter - P2A - L7Ae.

References

1.uang L, Lilley DMJ. 2018. The kink-turn in the structural biology of RNA. Q Rev Biophys 51: 1–32.doi:10.1017/S0033583518000033 2.Huang L, Lilley DMJ. 2013. The molecular recognition of kink turn structure by the L7Ae class of proteins. RNA 19: 1703–1710.doi:10.1261/rna.041517.113





Sequence and Features