Revision as of 11:51, 12 October 2023

stem-loop test

Introduction

In synthetic biology, it's common to integrate multiple heterologous genes into genetic circuits. Conventional approaches often utilize a polycistron system, where several genes are regulated under a single promoter. However, this can lead to reduced expression of downstream genes^[1].

In ribozyme-assisted polycistronic co-expression system (pRAP)^[2], the RNA sequence of ribozyme between coding sequences (CDSs) can conduct self-cleavage and convert polycistron into mono-cistrons in vivo. Self-interaction of the polycistron can be avoided and each mono-cistron can initiate translation with efficiency.

Figure 1. Principle of pRAP System.

In the pRAP system stem-loop is a key regulatory element that affects protein concentration by regulating the rate of mRNA degradation ^[3]. By regulating the intensity of stem-loop, we can control the expression of proteins.

This year, we developed a quantitive pRAP system design software, and we also construct this composite part BBa_K4765129 (stem-loop test) to verify that we can control protein expression by changing stem-loop.

This composite includes T7 promoter, lac promoter, stayGold, Twister P1 (self-cleaving ribozyme), mScarlet, and T7 terminator, which is a stem-loop-deleted version of BBa_K4765120. We inserted different stem-loops between stayGold and Twister P1, and compared the red-green fluorescence intensity ratio to assess the stem-loop's ability to prevent mRNA degradation.

Figure 2. Biobricks in BBa_K4765129.

Usage and Biology

We use this composite part to test the following stem-loops' ability to prevent mRNA degradation.

nsl:     5-AAACACCCACCACAAUUUCCACCGUUU UUUGU-3
liu2023: 5-AAACACCCACCACAAUUUCCACCGUUU CCCGACGCUUCGGCGUCGGG UUUGU-3
new2:    5-AAACACCCACCACAAUUUCCACCGUUU CCCCGUCGGCUGCU UUUGU-3
new6:    5-AAACACCCACCACAAUUUCCACCGUUU AGACGCUCGGCGUCCU UUUGU-3
new10:   5-AAACACCCACCACAAUUUCCACCGUUU ACUGGGGGGAUCGAGGUCUUU UUUGU-3
old2:    5-AAACACCCACCACAAUUUCCACCGUUU GCCGAUCGGGU UUUGU-3
old6:    5-AAACACCCACCACAAUUUCCACCGUUU AGACGCUCGGCGUCCU UUUGU-3
old10:   5-AAACACCCACCACAAUUUCCACCGUUU GGCGGCGCUACAGCGUCGU UUUGU-3

Characterization

Sequencing Map

Figure 3. Sequencing result of nsl (no stem-loop before Twister ribozyme cleavage site). Sanger sequencing verified that we have removed the stem-loop before ribozyme sequence, from BBa_K4765120. We also construct plasmids with stem-loop new2, new6, new10, bad2, bad6, bad10, all of which were designed by our Software RAP, and fully characterized using functional assays.

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Sequence and Features

Reference

1. ↑ Kim, K.-J., Kim, H.-E., Lee, K.-H., Han, W., Yi, M.-J., Jeong, J., & Oh, B.-H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Science: A Publication of the Protein Society, 13(6), 1698–1703. https://doi.org/10.1110/ps.04644504
2. ↑ Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143. https://doi.org/10.1021/acssynbio.2c00416
3. ↑ Newbury, S. F., Smith, N. H., & Higgins, C. F. (1987). Differential mRNA stability controls relative gene expression within a polycistronic operon. Cell, 51(6), 1131–1143. https://doi.org/10.1016/0092-8674(87)90599-x