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| | <partinfo>BBa_K2259092 short</partinfo> | | <partinfo>BBa_K2259092 short</partinfo> |
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| − | This is the backbone base vector 2.0, which contains no insert. See [[Part:BBa_K2259081 BBa_K2259081]] for the full vector. | + | This is the backbone base vector 2.0, which contains no insert. See [[Part:BBa_K2259081]] for the full vector. |
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| − | Engineering an improved, functional base vector 2.0 was crucial for SynORI framework, because building a synthetic origin of replication required an empty biobrick site and no origin of replication in backbone. One can then replace the pUC origin of replication to SynORI system parts. Once the modular SynORI system is built, it can be transfered to another plasmid location and biobricks are then free to use for other projects required. | + | Engineering an improved, functional base vector 2.0 is crucial for SynORI framework. |
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| − | See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
| + | Building a modular a synthetic origin of replication is easiest when working in biobrick region. But for synthetic ORI this is only possible if there is no other origin of replication in the rest of the vector. Base vector 2.0 provides this, as it has its pUC replicon in biobrick site. |
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| | + | One can then replace the pUC origin of replication to SynORI system parts. Once the modular SynORI system is built, it can be transfered to another plasmid location and biobricks are then free to use for any task. |
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K2259092 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K2259092 parameters</partinfo>
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| − | <!-- -->
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| − | __TOC__
| + | <span class='h3bb'>Sequence and Features</span> |
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| − | =Introduction=
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| − | ==Biology==
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| − | ===ColE1 plasmid replication overview===
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| − | [[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]]
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| − | <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).
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| − | <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).
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| − | 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.
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| − | Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
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| − | ==Usage with SynORI (Framework for multi-plasmid systems)==
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| | ===About SynORI=== | | ===About SynORI=== |
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| | 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]! | | 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]! |
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| − | ===Regulative RNA II molecule in SynORI===
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| − | RNA II gene is foundational and central biobrick of SynORI system, and by far the only one that is mandatory for framework to run.
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| − | The two main functions of RNA II is as follows:
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| − | # Initiating plasmid replication
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| − | # Interacting with RNA I of specific plasmid group [[#Specific RNA II versions in multi-plasmid systems|(See below)]]
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| − | === RNA II and RNA I in the engineering of unique plasmid groups for multi-plasmid system===
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| − | 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.
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| − | <b>For example</b> if there are two plasmid groups in a cell - A and B - RNA II of A group
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| − | would only interact with RNA I A, and not RNA I B.
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| − | [[Image:RnainteractionIII.png|center|500px|thumb|<b>Figure 1. </b> RNA I AND II group interaction example]]
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| − | 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).
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| − | ===Origin of RNA II biobrick===
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| − | If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?
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| − | 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>.
| + | See [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI). |
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| − | <Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
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| | =Characterization of RNA II (Vilnius-Lithuania 2017)= | | =Characterization of RNA II (Vilnius-Lithuania 2017)= |
Engineering an improved, functional base vector 2.0 is crucial for SynORI framework.
Building a modular a synthetic origin of replication is easiest when working in biobrick region. But for synthetic ORI this is only possible if there is no other origin of replication in the rest of the vector. Base vector 2.0 provides this, as it has its pUC replicon in biobrick site.
One can then replace the pUC origin of replication to SynORI system parts. Once the modular SynORI system is built, it can be transfered to another plasmid location and biobricks are then free to use for any task.
