Zoepin commented 3 years ago

An interesting point was raised by Aya: how can we be sure the plasmid contained in the mother cell will be transferred to the minicell? It was then suggested that we look at the iGEM Vilnius-Lithuania 2017 iGEM project on plasmid copy number control and partitioning system.

SynOri

Vilnius-Lithuania 2017 iGEM http://2017.igem.org/Team:Vilnius-Lithuania

Description

SynORI stands for synthetic origin of replication. It is a framework designed to make working with single and multi-plasmid systems precise, easy and on top of that - more functional.

The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:

Selecting the number of plasmid groups
Choosing the copy number of each group
Picking the type of copy number control (specific to one group or regulating all of them at once).

The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low copy number plasmid groups are not lost during the division.

Design and results

Determining the plasmid copy number (PCN)

They used absolute quantitative PCR with 2 sets of primers:

1 for the plasmid Ori => since the Ori is different for each plasmid group, to distinguish them
1 for a chromosomal gene (dxs). They divide the total PCN by the cell number => ratio of these 2 genes copy number gives PCN per cell.

Plasmid copy number control

Based on re-engineered ColE1 origin of replication.

Explanation on how the ColE1 replicon works: http://2017.igem.org/Team:Vilnius-Lithuania/Design

The plasmid replication is controlled by 2 sequences: RNAII that will act as an initiator and RNAII which is the replication inhibitor. Once transcribed, the RNAII acts as a primer for DNA polymerase. If transcribed, the RNAI forms a complex with RNAII and inhibits transcription. (The animations in the link above are very helpful to understand the mechanism.) In order to control the plasmid copy number, they found a way to modify the RNAI promoter without changing the RNAII secondary structure.

Once the RNA I promoter was disabled in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit.

=> They are now able to control the plasmid copy number in a constitutive way. => They used a rhamnose dependent promoter to build an inducible copy number system, where the rhamnose promoter controls the transcription of RNAI.

If using an Anderson promoter, they created a model to determine which promoter to use in order the obtain the precise plasmid copy number wanted. http://2017.igem.org/Team:Vilnius-Lithuania/Model

Multiple plasmid groups

Multi-plasmid framework would not be much without multiple plasmids. We have equipped our synthetic origin of replication with specific sequences to create unique plasmid groups.

They engineered different plasmid groups by adding unique, group specific sequences to RNA I and RNA II stem loops.

=>The engineered RNAII A and RNA II B were concluded to act as different replicon without inhibiting the replication of each other.

Global copy number regulation

Using the Rop protein to control every plasmid group at the same time. The Rop protein recognizes RNA secondary structures rather than unique DNA sequences. When Rop binds to secondary structures, it increases the binding affinity of RNA I and RNA II and consequently-replication inhibition. Therefore, whatever the sequences of RNA I and RNA II in the different plasmid groups, the plasmid replication can be controlled by Rop.

=> They showed that Rop protein with an Anderson promoter can reduce the copy number of multiple plasmids non-specifically.

Selection system

2 plasmid system

To address this problem a protein granting the resistance to aminoglycoside family antibiotics, called amino 3'-glycosyl phosphotransferase (APH(3')), was split into two subunits by Calvin M. Schmidt et al. They created a split antibiotic system where 1 plasmid expresses 1 sub-unit of the protein and another plasmid the other sub-unit.

4 plasmid system

2 toehold switches are activated by 2 different plasmids. Each toehold switch, when activated, allows the translation of 1 sub-unit of the resistance protein.

5 plasmids system - phage control

The fifth plasmid would house a transcription factor for the initiation of whole system.

Active partitioning system

SynORI framework provides an opportunity to have low copy plasmid groups, yet in order for them not to be lost during the cell division, there must be a mechanism that actively keeps plasmids in the cell.

They used a described pSC101 plasmid which contains a binding site for DNA gyrase. It has been showed that pSC101 plasmids with partial deletions of stability region have decreased supercoiling and are extremely unstable. This has led to the proposal that gyrase-generated negative supercoiling establishes a DNA conformation which enables plasmids to interact with specific E. coli structures and distribute them to daughter cells during cell division.

JulietteB-cri commented 3 years ago

I think having a 2 plasmids system could really work. Here is another idea proposed by Ariel:

ELK16 peptide

Ariel also proposed a system where the self-assembled peptide ELK16 ((LELELKLK)2 could bind to a DBD on the plasmid (on not on the chromosome) in order to have the plasmid on the pole of the mother cells and thus ensuring it would be in the minicells too.

Wu, W., Xing, L., Zhou, B. et al. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli. Microb Cell Fact 10, 9 (2011). https://doi.org/10.1186/1475-2859-10-9