View source for Part:BBa K2259086


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RNA I acts as a plasmid replication inhibitor. RNA I gene transcript's secondary structures, which consists of three stem loops, binds to RNA II (replication initiator) secondary structures and - if successful -  inhibits plasmid replication initiation. 

This part is a fully wild type ColE1 RNA I gene with its wild type promoter intact.


See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]

<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2259086 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2259086 parameters</partinfo>
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=Introduction=
==Biology==
===ColE1 plasmid replication overview===

[[Image:Cole1 horizontal cropped.png|center|frame|300px|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication]]



<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).<ref>Itoh, T. and Tomizawa, J. (1980). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proceedings of the National Academy of Sciences, 77(5), pp.2450-2454.</ref>

<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).<ref>Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.</ref>

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. <ref>Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.</ref>

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===
[[Image:synori.png|600px|center|]]
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 I molecule in SynORI===
The main goal of RNA I in the framework is group-specific control of copy number. Different plasmid copy numbers are achieved by changing RNA I concentration in the cell.

===Specific RNA I and RNA II versions in SynORI framework===

As RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this fact to our advantage in order to engineer different plasmid groups by adding unique, group-specific sequences to RNA I and RNA II stem loops.

 <b>For example</b> 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.

The inactivation and transfer of RNA I gene away from RNA II allow us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other.

Since there are three stem loops responsible for RNA I – RNA II interaction for each of the plasmid group we have decided to:

* Use two different unique sequences in the first two stem loops, in order to maximize same group specificity.
* For the third loop, we have decided to keep RNA II unchanged, and add either G/C mutations (GC type RNA I) or make RNA I completely non-complement to RNA II (NC type RNA I).

We did not want to introduce new specific sequences into the third loop of RNA II sequence. That is because according to literature <links> RNA II secondary structures at third loop structure are very sensitive to any mutations and has a high chance of ruining the replication initiation. Just because we chose not to interfere with the third loop of RNA II, we could not leave RNA I gene unchanged. If every group would have the fully compatible third loop, the background cross-group inhibition would be too large.

So now we have 5 different RNA II genes corresponding to groups A B C D and E.

Also, we have 10 different RNA I alternatives:
A, B, C, D, E with each having a version of either G/C or NC mutations.

So for example, if we have a part named RNA I (B-NC), it means: This RNA will only selectively regulate RNA II molecule by having specific B group sequences in first two stem loops. Also, in the third stem loop every nucleotide is not complementary to RNA II third loop.

These different plasmid groups (A-E) can then be co-maintained in a cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.


[[Image:RnainteractionIII.png|center|500px|thumb|<b>Figure 2. </b> RNA I AND II group interaction example]]

===Origin of RNA I biobrick===

[[File:4animacija.gif|centre|frame|200px|<b>Figure 3. </b> Changes introduced in RNA I promoter sequence also changes RNA II secondary structure]]

In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in the ColE1 origin of replication. That, however, was not a trivial task, as ColE1 ORI is an antisense system, which means that by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation (Find how this problem was solved at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]). This is the main reason why, in the SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.

=Characterization of RNA I (Vilnius-Lithuania 2017)=
==Interaction between RNA I and RNA II groups==
===Constitutive promoter===

Once the RNA I promoter was [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2259000#RNA_I_inactivation_in_wild_type_replicon disabled] in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit. We have discovered the sequence of wild type RNA I promoter by using PromoterHunter and removed it, thus creating a wild type RNA I gene [[part:BBa_K2259005]]. First, series of Anderson promoters were cloned next to the RNA I gene ([[part:BBa_K2259021]] (0.15 Anderson), [[part:BBa_K2259023]] (0.36 Anderson), [[part:BBa_K2259027]] (0.86 Anderson), [[part:BBa_K2259028]] (1.0 Anderson)) and then placed next to RNA II ([[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson), [[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson)).

[[File:RNA_I_anderson.png|thumb|centre|900px|<b>Figure 4. </b> RNA I and RNA II constructs, with RNA I constructs under different-strength Anderson promoters.]]

In theory (see “Modelling” at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]), lower-strength Anderson promoters should yield lower concentrations of RNA I, hence higher copy numbers of plasmids per cell.  Our constitutive copy number device experiment results prove it to be true in practice as well. The stronger Anderson promoter is used, the less copy number per cell we get. With the strongest Anderson we get only 21+-6.84 plasmids per cell. 

Worth to mention is that the closest to wild type ColE1 replicon is the 0.86 strength Anderson promoter ([[Part:BBa_J23102]]), measured by copy number alone. 

We can state with certainty that we are now able to control the plasmid copy number in a constitutive manner, and we simply call it the SynORI constitutive copy number device.

===Inducible promoter===

We wanted to move one step further and try to build an inducible copy number system. We first had to make sure that at least part of our construct is well characterized and to do so we chose the Rhamnose promoter from the biobrick registry ([[Part:BBa_K914003]])

For this experiment we have built a Rhamnose promoter and RNA I construct [[part:BBa_K2259065]] and then cloned this construct next to RNA II [[part:BBa_K2259091]]. We have used different concentration of Rhamnose in our media in order to see if this approach was possible and if so, to figure out the dependency between the plasmid copy number and rhamnose concentration.

[[File:Rha_rnr.png|thumb|centre|900px|<b>Figure 5. </b> RNA I and RNA II constructs, with RNA I gene being under the Rhamnose promoter, inducided by different rhamnose concentrations.]]

The first thing we noticed was that Rhamnose promoter was very strong in terms of plasmid copy number reduction. It was also considerably leaky (promoter can be enabled even without any inducer). At zero induction there were approximately only 9 plasmids per cell and at 1 percent induction the number dropped to approximately 1 plasmid per cell. 

RNA I rhamnose-induced promoter seemed to be working well, with higher concentrations of inductor giving lower plasmid copy number.

We called it the SynORI copy number induction device.

So now when we can flexibly control the copy number of a plasmids, the only question is - what will come next?


==References==
<references />