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J23100-ALeaderT-BCD

A regulator containing a bi-cistron design.

Sequence and Features

Background

BCD (bi-cistronic design) is a method recently developed for precise expression (Mutalik V K, et al, 2013). It refers to a nested mRNA motif, initiating the translation of a short leader peptide from a defined SD motif (Figure 1). The cistron 2 overlaps by a single base with the start codon of the downsteam cistron 3 via a -1 frame shift (e.g., TAATG). Translation of the cistron 3 is initiated from a second SD motif that is entirely embedded within the coding sequence of the leader peptide. Because the ribosomes slip though the cistron 2, the SD3 in cistron 2 is not interfered by surrounding RNA, thereby initiating stable expression. This expression is only related to the RNA transcriptional rate and RBS strength of SD3(Jia X, et al, 2013.).

Design

In our modelling process, we predicted the conformation B of ALeaderT as a functional terminator, and such suggestion is also inferred by dose-response curve. Furthermore, that termination could be inhibited by kanamycin. To validate that hypothesis and then quantify the dose-termination curve, we combined the AleaderT and a standard BCD variant to form a tri-cistron design (Figure 1).

We replaced SD2 to the first ATG in BCD with ALeaderT, so that the transcription can be measured by the fluorescence test.

If ALeaderT conformation B is a functional terminator, there are two possible results caused by the stem loop formation, the extension and the termination. In the case of the extension and in the conformation C, the translation from SD2 would be terminated by TAA in sequence TAATG, while translation from SD3 would be initiated by ATG in that same sequence, resulting in a precise mRFP1 expression. In the conformation A, mRFP1 is produced as well, less precisely. Only in the case of the termination there is no translation of reporter (Figure 2).

Quantitative measurement Data

The mRFP1 expression is low without or at low Kanamycin concentration. Increasing the Kanamycin can increasing the fluorescence. Since the mRFP1 expression can only be initiated by SD3 as previous stated, and SD3 initiation is related to mRNA concentration and its strength, it is confirmed that ALeaderT has terminator function, and that ALeaderT caused termination is inhibited by Kanamycin (Figure 3).\

Reference

[1] Mutalik V K, Guimaraes J C, Cambray G, et al. Precise and reliable gene expression via standard transcription and translation initiation elements[J]. Nature methods, 2013, 10(4): 354-360. [2] Jia X, Zhang J, Sun W, et al. Riboswitch Control of Aminoglycoside Antibiotic Resistance[J]. Cell, 2013, 152(1): 68-81.