Revision as of 08:52, 31 October 2017

SynORI base vector 2.0 insert

This composite part is designed to replace the base vector's Part:BBa_I51020 insert which is no longer available because of the ccdB part containing construct withdrawal.

Vector backbone which does not contain this insert can be [found here.]

These combined sequences result in the new Base vector 2.0, which instead of ccdB poison uses blue white screening to select for correct colonies when building a custom plasmid.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 462
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Introduction

Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

SynORI is a framework for multi-plasmid systems created by Vilnius-Lithuania 2017 which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!

Regulative RNA II molecule in SynORI

RNA II gene is foundational and central biobrick of SynORI system, and by far the only one that is mandatory for framework to run. The two main functions of RNA II is as follows:

Initiating plasmid replication
Interacting with RNA I of specific plasmid group (See below)

RNA II and RNA I in the engineering of unique plasmid groups for multi-plasmid system

RNA II molecule interacts with inhibitory RNA I molecule with three secondary structure RNA stem loops. In order to create plasmid groups with independent copy number control, one group's RNA II molecule must interact only with the same group's RNA I molecule.

For example if there are two plasmid groups in a cell - A and B - RNA II of A group
would only interact with RNA I A, and not RNA I B.

Figure 1. RNA I AND II group interaction example

See the Design section or [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).

Characterization of RNA II (Vilnius-Lithuania 2017)

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2259093 short</partinfo>
-RNA II acts as a plasmid replication initiatior. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the plasmids DNA. This RNA-DNA hybrid is attacked by RNase H, which cleaves the RNA strand, exposing a 3' hydroxyl group. This allows the extension of the leading strand by DNA polymerase I and consequently, the start of plasmid replication.
+This composite part is designed to replace the base vector's [[Part:BBa_I51020]] insert which is no longer available because of the ccdB part containing construct withdrawal.
-*Caution! <B>RNA II (Group A)</b> indicates that this plasmid only interacts with regulatory <B>RNA I (Group A)</b> <LINK TO RNA I A> from SynORI (framework for multiplasmid systems) collection and is stable when placed with other SynORI plasmid groups. RNA II A will not be regulated with RNA I from another group!
+Vector backbone which does not contain this insert can be [[https://parts.igem.org/wiki/index.php?title=Part:BBa_K2259092 found here.]]
-See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
+These combined sequences result in the new Base vector 2.0, which instead of ccdB poison uses blue white screening to select for correct colonies when building a custom plasmid.
 <span class='h3bb'>Sequence and Features</span>
@@ Line 24: / Line 23: @@
 =Introduction=
-==Biology==
-===ColE1 plasmid replication overview===
-[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]]
-<b>ColE1-type plasmid replication begins with synthesis of plasmid encoded RNA II</b> (also called primer transcript) by RNA polymerase which initiates transcription at a site 555bp upstream of origin of replication. The RNA transcript forms a RNA - DNA hybrid with template DNA near the origin of replication. Hybridized RNA is then cleaved at the replication origin by RNAse H and serves as a primer for DNA synthesis by DNA polymerase I (Figure 1. A).
-<b>Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I </b>. Synthesis of RNA I starts 445 bp upstream of the replication origin and proceeds in the direction opposite to that of RNA II synthesis, and terminates near the RNA II transcription initiation site. <b>RNA I binds to RNA II</b> and thereby prevents formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).
-For RNA I to inhibit primer formation, it must bind before the nascent RNA II transcript extends to the replication origin. Consequently, the concentration of RNA I and the rate of binding of RNA I to RNA II is critical for regulation of primer formation and thus for plasmid replication.
-Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
 ==Usage with SynORI (Framework for multi-plasmid systems)==
@@ Line 60: / Line 48: @@
 See the [https://parts.igem.org/Part:BBa_K2259000:Design Design] section or [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).
-===Origin of RNA II biobrick===
-If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?
-In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in ColE1 origin of replication. That, however, was not a trivial task, because by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation. This is the main reason why, in SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.
-<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
 =Characterization of RNA II (Vilnius-Lithuania 2017)=

Difference between revisions of "Part:BBa K2259093"