Difference between revisions of "Part:BBa K2259067"
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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)]] | [[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>ColE1-type plasmid replication begins with the 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 | + | <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 the 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. | 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. | ||
| − | + | The 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)== | ==Usage with SynORI (Framework for multi-plasmid systems)== | ||
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===This device in SynORI=== | ===This device in SynORI=== | ||
| − | This is a constitutive copy number device which sets a specific copy number for a plasmid. These constitutive devices can be used with different | + | This is a constitutive copy number device which sets a specific copy number for a plasmid. These constitutive devices can be used with different Anderson promoters to select a different copy number. |
Devices from the same series that have different Anderson promoters: [[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson),[[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson). | Devices from the same series that have different Anderson promoters: [[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson),[[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson). | ||
Revision as of 15:52, 31 October 2017
SynORI constitutive plasmid copy number device (0.15)
This device is a fully functional synthetic origin of replication that sets a constitutive copy number to a plasmid. Different concentrations of RNA I gene provide a different copy number of a plasmid.
Devices from the same series that have different Anderson promoters: part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson), part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson).
See how this part fits into the whole SynORI framework by pressing here!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 685
Illegal NheI site found at 708 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
Biology
ColE1 plasmid replication overview
ColE1-type plasmid replication begins with the synthesis of plasmid encoded RNA II (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).
Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I . 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. RNA I binds to RNA II and thereby prevents the 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.
The 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)
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]!
This device in SynORI
This is a constitutive copy number device which sets a specific copy number for a plasmid. These constitutive devices can be used with different Anderson promoters to select a different copy number.
Devices from the same series that have different Anderson promoters: part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson),part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson).
See the [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).
Further details
For more background information and indepth insight on this part's design please see the individual part pages of part:BBa_K2259000 and part:BBa_K2259005.