Difference between revisions of "Part:BBa K864018"
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<partinfo>BBa_K864018 short</partinfo>
 
<partinfo>BBa_K864018 short</partinfo>
  
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pSB8K15 is a BioBrick standard vector with low copy pSC101ts replication origin <partinfo>BBa_K864050</partinfo> (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30’C but not at at 42’C [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
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pSB8K15 is a BioBrick standard vector with low copy pSC101ts replication origin <partinfo>BBa_K864051</partinfo> (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30ºC but not at at 42ºC [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
  
 
===Usage and Biology===
 
===Usage and Biology===
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'''FLP recombinase'''
 
'''FLP recombinase'''
  
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The pSB8X15 series are especially suitable for carrying the FLP recombinase, since plasmids can be removed by incubation at 42°C.  
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The pSB8X15 series are especially suitable for carrying systems where a temporary expression is desired, such as the Lambda Red recombineering system or recombinases like Cre or FLP, since the plasmids can be removed afterwards by incubation at 42°C.  
  
 
'''Origin switching'''
 
'''Origin switching'''
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'''More reliable recombineering'''
 
'''More reliable recombineering'''
  
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A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C.  
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A problem when doing recombineering based chromosomal intergration is that some clones may take up the plasmid template instead of recombining the PCR fragment into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C.  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 00:18, 30 September 2012

Low copy BioBrick temperature sensitive standard vector

pSB8K15 is a BioBrick standard vector with low copy pSC101ts replication origin BBa_K864051 (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30ºC but not at at 42ºC [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.

Usage and Biology

This part is available as pSB8K15-pUCori-redq. This facilitates fast growth at 37° C, high plasmid yields and fast red color expression on transformation plates.

FLP recombinase

The pSB8X15 series are especially suitable for carrying systems where a temporary expression is desired, such as the Lambda Red recombineering system or recombinases like Cre or FLP, since the plasmids can be removed afterwards by incubation at 42°C.

Origin switching

Easy ori replacement is possible in the pSB8x15 backbones. The plasmid can have the pSC101ts ori cut out with NheI and MluI and another ori ligated in. To remove any religated pSC101ts, the transformants can be grown at 42° C.

More reliable recombineering

A problem when doing recombineering based chromosomal intergration is that some clones may take up the plasmid template instead of recombining the PCR fragment into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3642
    Illegal NheI site found at 2425
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3648
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3642
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3642
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3642
    Plasmid lacks a suffix.
    Illegal XbaI site found at 3657
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 2319
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.