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Present Challenges of
Experimenting With Plasmid Systems

1. 1.

Plasmid Replication

Origin of replication (Ori) is a sequence where the plasmid replication begins. Plasmids with similar Ori sites use the same replication machinery.

Problem #1

1. 2.

Unbalanced Replication

Replication machinery does not discriminate between similar Ori sites, therefore smaller plasmids are replicated faster and rapidly outgrow larger plasmids, which leads to uneven plasmid ratios in a cell.

Problem #1

1. 3.

Plasmid Loss

This is even more likely to occur when some plasmids are low copy number. When the cell divides, low copy plasmids tend to be lost in next generations.

Problem #1

2. 1.

Plasmid Incompatibility

This inability to stably co-maintain different plasmids over a number of generations in the same bacterial cell line is called plasmid incompatibility. One needs to use different types of Ori to avoid this trouble.

Problem #2

2. 2.

Plasmid Incompatibility

However, it often causes trouble due to limited Ori selection options or the lack of good combination of Ori and plasmid's copy number. Imagine you require a system consisting of two - low and high copy number plasmids. Try picking them from the list.

Problem #2

2. 3.

Fixed Copy Number

Currently no handy method of changing the copy number of particular plasmid exists. It must be either low, or medium, or high.

Problem #2

2. 4.

Deficient Responsivity

There is also no mechanism to adjust the copy number when so is required in any specific situation, for example: incoming cellular signals or special patterns.