Difference between revisions of "Part:BBa K4166005"

 
(→‎Plasmid Copy Number and Expression - Alma 2022)
 
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Runway plasmid replication
 
Runway plasmid replication
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== Plasmid Copy Number and Expression - Alma 2022 ==
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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).
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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:
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Comparison of qPCR data. Original (top) and mutant (bottom):
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[[File:T--Alma--wtpcn.png|650px|center]]
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[[File:T--Alma--camp8pcn.png|650px|center]]
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Based on several methods, we estimated the following ratio of plasmid to genomic DNA in a cell:
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J04450: 76
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K4166002: 107 (Lin-Chao A715G)
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K4166004: 307 (Camp +1)
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K4166005: 381 (Camp +8)
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K4166006: 133 (Camp +8 and -11)
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K4166007: 153 (Camp -11)
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K4166008: 194 (Camp +13)
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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=)
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[[File:T--Alma--gel_005.png|350px|right]]
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[[File:T--Alma--pcnPlate.png|350px|left]]
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From the intensity of these bands we estimate the copy number of this origin at ~387 copies/cell.
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We found that this plasmid both increases copy number and expression.
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<!-- Add more about the biology of this part here
 
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Latest revision as of 01:05, 14 October 2022


Camp +8 mutation.

Runway plasmid replication

Plasmid Copy Number and Expression - Alma 2022

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:

Comparison of qPCR data. Original (top) and mutant (bottom):

T--Alma--wtpcn.png
T--Alma--camp8pcn.png

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

J04450: 76 K4166002: 107 (Lin-Chao A715G) 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=)


T--Alma--gel 005.png
T--Alma--pcnPlate.png

From the intensity of these bands we estimate the copy number of this origin at ~387 copies/cell. We found that this plasmid both increases copy number and expression.


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.