Part:BBa_K4166002

Low copy number reversion

Helps revert low copy number of the DNA.

High copy number of pUC plasmids results from a single point mutation in the replication primer.

https://doi.org/10.1111/j.1365-2958.1992.tb02206.

Alma 2022 - Plasmid Copy Number Determination

This year, we planned two improvements to the commonly used part, J04450. To better control the sometimes leaky expression, or to maximize expression, we created mutants of the origin sequence present on all pSB1C3 backbones. We introduced mutations +1, +8, -11, -13 from Camps et al, 2010, and the A715G reversion mutant from Lin-Chao et al, 1992 (turning this back into pBR322, instead of pUC19).

We collected plasmid copy number information using a qPCR protocol, and different approaches to gel quantitation (such as those found in Lin-Chao et al 1992 (and the variation present on the Barrick lab protocols page), Pushnova et al 2000. Here are some representative results:

Original (top) versus Mutant (bottom)

Based on several methods, we estimated the following ratio of plasmid to genomic DNA in a cell:

J04450: 76 K4166002: 107 (Lin-Chao A715G) *** In the above experiment, it was as low as 7! K4166004: 307 (Camp +1) K4166005: 381 (Camp +8) K4166006: 133 (Camp +8 and -11) K4166007: 153 (Camp -11) K4166008: 194 (Camp +13)

In exponential cells, there is typically more than one copy of the genome present per cell, since the cells are actively replicating. Therefore, we would except to double or triple the above numbers to get a true plasmid copy number per cell. (This is based on the data at found from the BIONUMB3R5 database: https://bionumbers.hms.harvard.edu/bionumber.aspx?id=106438&ver=2&trm=Escherichia+Coli+Genome&org=)

We also observed the following expression pattern:

This indicates that there is similar or less expression when the plasmid copy number is decreased.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.