Revision as of 11:51, 12 October 2023

stem-loop test contributed by Fudan iGEM 2023

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.

==

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1389
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 700
Illegal BsaI.rc site found at 720

Reference

↑ 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
↑ 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
↑ 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

[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

[1]

[2]

[3]

@@ Line 7: / Line 7: @@
 ===Introduction===
-This year we further improved the ribozyme-assisted polycistronic co-expression system (pRAP).
-The major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter<ref>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</ref>. Compared with multiple promoter system, in pRAP, self-interaction of the polycistron can be avoid and each cistron can initiate translation with comparable efficiency. In pRAP system , the RNA sequences of ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons ''in vivo''.
+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<ref>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</ref>.<br>
-Stem-loop is a key regulatory element in pRAP, it affects protein concentration by regulating the rate of mRNA degradation. By regulating the intensity of stem-loop, we can control the expression of proteins.
-This compoiste part is a stem-loop-deleted version of [https://parts.igem.org/Part:BBa_K4765120 BBa_K4765120], which includes stayGold and mScarlet. Its red-green fluorescence intensity ratio can be compared with [https://parts.igem.org/Part:BBa_K4765120 BBa_K4765120] to assess the stem-loop's ability to prevent mRNA degradation.
+In ribozyme-assisted polycistronic co-expression system (pRAP)<ref>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</ref>, 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.
+{|
+| <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/1291.png" alt="contributed by Fudan iGEM 2023"></html>
+|-
+| '''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 <ref>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</ref>. By regulating the intensity of stem-loop, we can control the expression of proteins. <br>
+This year, we developed a quantitive pRAP system design [https://2023.igem.wiki/fudan/software/#overview 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. <br>
+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 [https://parts.igem.org/Part:BBa_K4765120 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 [https://2023.igem.wiki/fudan/software/#assumption-1-2 mRNA degradation].
+{|
+| <html><img style="width:300px" src="https://static.igem.wiki/teams/4765/wiki/results-wyj/1292.png" alt="contributed by Fudan iGEM 2023"></html>
+|-
+| '''Figure 2. Biobricks in BBa_K4765129.'''
+|}
 ===Usage and Biology===
-We use this composite part to test the following stem-loops'<ref>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</ref> ability to prevent mRNA degradation.
+We use this composite part to test the following stem-loops' ability to prevent mRNA degradation.
 <pre>nsl:     5-AAACACCCACCACAAUUUCCACCGUUU UUUGU-3
 liu2023: 5-AAACACCCACCACAAUUUCCACCGUUU CCCGACGCUUCGGCGUCGGG UUUGU-3
@@ Line 27: / Line 42: @@
 ===Characterization===
-====Sequencing map====
+====Sequencing Map====
 {|
 | <html><img style="width:640px" src="https://static.igem.wiki/teams/4765/wiki/zsl/stem-loop-test-sequence.png" alt="contributed by Fudan iGEM 2023"></html>
 |-
-| '''Figure 1. Sequencing result of nsl (no stem-loop before Twister ribozyme cleavage site).'''<br>Sanger sequencing verified that we have removed the stem-loop before ribozyme sequence, from [https://parts.igem.org/Part:BBa_K4765120 BBa_K4765120]. We also construct plasmids with stem-loop new2, new6, new10, bad2, bad6, bad10, all of which were designed by our [https://2023.igem.wiki/fudan/software Software RAP], and fully characterized using functional assays.
+| '''Figure 3. Sequencing result of nsl (no stem-loop before Twister ribozyme cleavage site).'''<br>Sanger sequencing verified that we have removed the stem-loop before ribozyme sequence, from [https://parts.igem.org/Part:BBa_K4765120 BBa_K4765120]. We also construct plasmids with stem-loop new2, new6, new10, bad2, bad6, bad10, all of which were designed by our [https://2023.igem.wiki/fudan/software Software RAP], and fully characterized using functional assays.
 |}
+====
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K4765129 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display

Difference between revisions of "Part:BBa K4765129"

Revision as of 11:51, 12 October 2023

Contents

Introduction

Usage and Biology

Characterization

Sequencing Map

==

Reference