Difference between revisions of "Part:BBa K4579008"

Revision as of 22:02, 5 October 2023

CvaC15 - Signal peptide

Introduction

The 2023 UT Austin iGEM Team’s Parts Collection includes a multitude of parts necessary for engineering bacteria to secrete microcins, a type of small antimicrobial peptide. Specifically, our team has designed parts that allow us to engineer a modular Biobrick-friendly version of an existing two-plasmid microcin secretion system1 that secretes putative novel microcins predicted by bioinformatics analysis.2 The first plasmid—the ‘microcin’ plasmid—contains the microcin and a signal peptide, while the second plasmid—the ‘secretion system’ plasmid—contains genes for two proteins of the E. coli microcin V type I secretion system (T1SS) machinery collectively referred to as CvaAB. Our parts can be easily assembled into transcriptional units to express any of our current 13 novel microcins (and potentially other small peptides1) either constitutively or under inducible control.

In our assembly schema, each basic part begins and ends with a distinct sequence of 4 nucleotides according to the part type derived from the syntax of the Bee Toolkit (BTK)3: Promoters, whether inducible or constitutive, are designed to function as Type 2 parts. The signal peptide is designed to function as a Type 3p part, while microcin or [microcin + immunity protein] parts are designed to function as Type 3q parts. Together, a Type 3p and Type 3q part form Type 3 parts in the BTK syntax. Our team designed the overhangs to connect Type 3p to Type 3q parts, and these part types are not present in the original Bee Toolkit. Terminators and [terminator + inducer-regulated transcription factor] parts are designed to function as Type 4 parts.

The various parts we have created can be assembled into microcin plasmid constructs with any other parts following the BTK syntax, allowing for the creation of flexible and modular designs by future iGEM teams that choose to work with microcins, secretion systems, or Golden Gate Assembly constructs. Additionally, we created a CvaAB part to allow for the recreation of the secretion system plasmid under a different promoter, origin, or selective marker depending on the needs at hand.

Characterization

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4579008 short</partinfo>
-We chose to design the CvaC15 as its own part rather than include it with the microcin in order to ensure that (1) any microcin, regardless of what bacterium it originated from, would be secreted by the E. coli T1SS encoded by the Davies Lab secretion system plasmid, and (2) the secretion system does not need to be changed for each microcin. All known microcins have a signal peptide sequence, but this can differ from microcin to microcin depending on the specific variants of the secretion system genes found within the genome of the bacterium from which the microcin originates. As such, by designing the signal peptide as its own assembly part, any microcin or other small peptide can be swapped in and out of the system with the confidence that it will always be fused to the T1SS-compatible signal peptide when translated. The CvaC15 signal peptide was nested in a high copy entry vector with a ColE1 origin of replication with chloramphenicol resistance.
+===Introduction===
-https://static.igem.wiki/teams/4579/wiki/aria-welch.jpg
+The 2023 UT Austin iGEM Team’s Parts Collection includes a multitude of parts necessary for engineering bacteria to secrete microcins, a type of small antimicrobial peptide. Specifically, our team has designed parts that allow us to engineer a modular Biobrick-friendly version of an existing two-plasmid microcin secretion system1 that secretes putative novel microcins predicted by bioinformatics analysis.2 The first plasmid—the ‘microcin’ plasmid—contains the microcin and a signal peptide, while the second plasmid—the ‘secretion system’ plasmid—contains genes for two proteins of the E. coli microcin V type I secretion system (T1SS) machinery collectively referred to as CvaAB. Our parts can be easily assembled into transcriptional units to express any of our current 13 novel microcins (and potentially other small peptides1) either constitutively or under inducible control.
+In our assembly schema, each basic part begins and ends with a distinct sequence of 4 nucleotides according to the part type derived from the syntax of the Bee Toolkit (BTK)3:
+Promoters, whether inducible or constitutive, are designed to function as Type 2 parts.
+The signal peptide is designed to function as a Type 3p part, while microcin or [microcin + immunity protein] parts are designed to function as Type 3q parts. Together, a Type 3p and Type 3q part form Type 3 parts in the BTK syntax.
+Our team designed the overhangs to connect Type 3p to Type 3q parts, and these part types are not present in the original Bee Toolkit.
+Terminators and [terminator + inducer-regulated transcription factor] parts are designed to function as Type 4 parts.
+https://static.igem.wiki/teams/4579/wiki/parts-collection-images/parts-collection-images/parts-collection-images/screenshot-2023-10-05-at-4-56-35-pm.png
+The various parts we have created can be assembled into microcin plasmid constructs with any other parts following the BTK syntax, allowing for the creation of flexible and modular designs by future iGEM teams that choose to work with microcins, secretion systems, or Golden Gate Assembly constructs. Additionally, we created a CvaAB part to allow for the recreation of the secretion system plasmid under a different promoter, origin, or selective marker depending on the needs at hand.
+===Characterization===
 <!-- Add more about the biology of this part here