Plasmid backbones/Library screening

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Plasmid backbones/Library screening
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Or get some help on plasmid backbones.


Promoter libraries • Protein coding sequence libraries • RBS & protein coding sequence libraries

One approach to designing new parts for the Registry is to

  1. generate libraries of randomized part sequences through use of random oligo's or error-prone PCR
  2. screen the library for functional parts via FACS or some type of colony assay
  3. isolate and sequence functional parts
  4. submit new functional parts to the Registry for reuse and improvement!

Using BioBrick® standard assembly to screen part libraries is not ideal, because once the library has been assembled with other parts (for example, a reporter to facilitate screening of the library), individual part library members cannot be easily extracted again via restriction digest. Hence, several plasmid backbones have been designed to facilitate screening of part libraries.

Note that these library screening plasmid backbones do NOT comply to the BioBrick® assembly standard and should only be used to screen part libraries.

The unique feature of these library screening plasmid backbones is that individual library members can be extracted from the library screening plasmid backbone via restriction digest to obtain a BioBrick® part in either prefix or suffix format, depending on the library plasmid backbone used. But again, the library screening plasmid backbones do not comply to the BioBrick® assembly standard and should be used with caution!

JCA Photo.png
 Chris Anderson, a professor of bioengineering at UC Berkeley, constructed the promoter and protein coding sequence library screening plasmid backbones, along with the ribosome binding site + protein coding sequence library backbone BBa_J23006.
ZhanJian.png
 Zhan Jian, as a member of the 2007 University of Science and Technology of China iGEM team, constructed the ribosome binding site + protein coding sequence library backbone BBa_I732950 and BBa_I732952.

Screening promoter libraries

Are you interested in making a library of promoters with different strengths? Then you might consider using this promoter library screening plasmid backbone BBa_J61002.
  1. Design your promoter library with a BioBrick® prefix and BioBrick® suffix
  2. Digest your library with EcoRI and SpeI
  3. Digest the promoter library screening plasmid backbone with EcoRI and SpeI
  4. Ligate the library into the promoter library screening plasmid backbone
  5. Screen the library and identify function promoter parts
  6. Extract functional promoter parts from the library via digest with EcoRI and SpeI for assembly into standard BioBrick® plasmid backbones.
PromoterLibrary.png


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Screening protein coding sequence libraries

Are you interested in making a protein coding sequence library? Then you might consider using one of these protein coding sequence library screening plasmid backbones.
  1. Design your protein coding sequence library with a BioBrick® prefix and BioBrick® suffix
  2. Digest your library with XbaI and PstI
  3. Digest the protein coding sequence library screening plasmid backbone with XbaI and PstI
  4. Ligate the library into the protein coding sequence library screening plasmid backbone
  5. Screen the library and identify functional protein coding sequence parts
  6. Extract functional protein coding sequence parts from the library via digest with XbaI and PstI for assembly into standard BioBrick® plasmid backbones.
CDSLibrary.png


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Screening ribosome binding sites & protein coding sequence libraries

Are you interested in making a library of composite parts of ribosome binding site plus protein coding sequence (RBS.CDS)? Then you might consider using a RBS.CDS library screening plasmid backbone.
  1. Design your RBS.CDS library with a BioBrick® prefix and BioBrick® suffix
  2. Digest your library with XbaI and PstI
  3. Digest the RBS.CDS library screening plasmid backbone with XbaI and PstI
  4. Ligate the library into the RBS.CDS library screening plasmid backbone
  5. Screen the library and identify functional RBS.CDS composite parts
  6. Extract functional RBS.CDS composite parts from the library via digest with XbaI and PstI for assembly into standard BioBrick® plasmid backbones.
RBSCDSLibrary.png


There are no parts for this table