Team:Amsterdam/Biobricks/Composite Parts

From 2011.igem.org

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*[http://partsregistry.org/Part:BBa_K538311 BBa_K538311]: pBAD + RBS + ''Cpn60'' + RBS + ''Cpn10''
*[http://partsregistry.org/Part:BBa_K538311 BBa_K538311]: pBAD + RBS + ''Cpn60'' + RBS + ''Cpn10''
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The four parts containing 2 RBSes and coding regions were designed like this, because ''Cpn10'' and ''Cpn60'' should be coexpressed for them to be functional, according to literature. Coexpression can be achieved by transforming ''E. coli'' with two plasmids, but we feel this operon-like brick structure is also an elegant solution.  [http://partsregistry.org/Part:BBa_K538210 BBa_K538210] should be functionally identical to [http://partsregistry.org/Part:BBa_K538211 BBa_K538211], as should [http://partsregistry.org/Part:BBa_K538310 BBa_K538310] and [http://partsregistry.org/Part:BBa_K538311 BBa_K538311] be. They only differ in the order of their coding regions, which resulted from us planning to assemble the operons by appending [http://partsregistry.org/Part:BBa_K538100 BBa_K538100] to [http://partsregistry.org/Part:BBa_K538201 BBa_K538201] and [http://partsregistry.org/Part:BBa_K538301 BBa_K538301], or by appending [http://partsregistry.org/Part:BBa_K538101 BBa_K538101] to [http://partsregistry.org/Part:BBa_K538200 BBa_K538200] and [http://partsregistry.org/Part:BBa_K538300 BBa_K538300].
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The four parts containing 2 RBSes and coding regions were designed the way they are, because ''Cpn10'' and ''Cpn60'' should be coexpressed for them to be functional, according to literature. Coexpression can be achieved by transforming ''E. coli'' with two plasmids, but we feel this operon-like brick structure is also an elegant solution.  [http://partsregistry.org/Part:BBa_K538210 BBa_K538210] should be functionally identical to [http://partsregistry.org/Part:BBa_K538211 BBa_K538211], as should [http://partsregistry.org/Part:BBa_K538310 BBa_K538310] and [http://partsregistry.org/Part:BBa_K538311 BBa_K538311] be. They only differ in the order of their coding regions, which resulted from us planning to assemble the operons by appending [http://partsregistry.org/Part:BBa_K538100 BBa_K538100] to [http://partsregistry.org/Part:BBa_K538201 BBa_K538201] and [http://partsregistry.org/Part:BBa_K538301 BBa_K538301], or by appending [http://partsregistry.org/Part:BBa_K538101 BBa_K538101] to [http://partsregistry.org/Part:BBa_K538200 BBa_K538200] and [http://partsregistry.org/Part:BBa_K538300 BBa_K538300].
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Even though we tried many different assembly protocols to attain complete CryoBricks, our progress was impaired by untracable technical difficulties. At the time of this writing, only two protein generators - the ''Cpn10'' and ''CspC'' generators under control of pLacI - are assembled and characterised, and because neither of them could be transferred to the pSB1C3 backbone succesfully, none of the CryoBrick generators are available from the registry for the time being.
Even though we tried many different assembly protocols to attain complete CryoBricks, our progress was impaired by untracable technical difficulties. At the time of this writing, only two protein generators - the ''Cpn10'' and ''CspC'' generators under control of pLacI - are assembled and characterised, and because neither of them could be transferred to the pSB1C3 backbone succesfully, none of the CryoBrick generators are available from the registry for the time being.

Revision as of 05:28, 21 September 2011

Composite Parts

Two types of composite parts were submitted to the registry: construction intermediates, and finalized CryoBricks. The construction intermediates contain either a promoter and an RBS, or an RBS and a coding region. The finalized CryoBricks are all protein generators with a classic PRC (Promoter, Ribosome binding site, Coding region) structure, except for 4 operon-like parts which are structured PRCRC. No terminators were used in any of our parts; termination of transcription relies on the terminators encoded in iGEM's standard plasmid backbones.

Construction Intermediates

The overview below lists all parts that were used as intermediates in the construction of our finalized CryoBrick protein generators.

  • [http://partsregistry.org/Part:BBa_K538100 BBa_K538100]: RBS + Cpn10
  • [http://partsregistry.org/Part:BBa_K538101 BBa_K538101]: RBS + Cpn60
  • [http://partsregistry.org/Part:BBa_K538102 BBa_K538102]: RBS + SheDnaK
  • [http://partsregistry.org/Part:BBa_K538103 BBa_K538103]: RBS + CspA
  • [http://partsregistry.org/Part:BBa_K538104 BBa_K538104]: RBS + CspC
  • [http://partsregistry.org/Part:BBa_K538120 BBa_K538120]: pLacI + RBS
  • [http://partsregistry.org/Part:BBa_K538130 BBa_K538130]: pBAD + RBS


By prepending an RBS to our coding regions and appending an RBS to our selected promoters, we enabled two different assembly pathways for the construction of the PRC protein generators: add a promoter to an RC part, or add a coding region to a PR part. Note that these promoter + RBS parts contain no bricks that weren't already available from the registry. Construction of all the parts listed above succeeded, but transferring it from the pSB1A3 backbone to the pSB1C3 backbone proved so difficult it only succeeded with ?????. As a result, the remaining intermediates are uneligible for submission to iGEM and won't be available from the registry.

Finalized CryoBricks

The purpose of our finalized CryoBricks is to enhance E. coli 's cold resistance, by expressing chaperones from psychrophillic bacteria. Information regarding how these bricks achieve this can be found on the pages of coding regions these protein generators comprise, or on this wiki's basic parts page. The following list contains all CryoBrick generators designed by our team:

  • [http://partsregistry.org/Part:BBa_K538200 BBa_K538200]: pLacI + RBS + Cpn10
  • [http://partsregistry.org/Part:BBa_K538201 BBa_K538201]: pLacI + RBS + Cpn60
  • [http://partsregistry.org/Part:BBa_K538202 BBa_K538202]: pLacI + RBS + SheDnaK
  • [http://partsregistry.org/Part:BBa_K538203 BBa_K538203]: pLacI + RBS + CspA
  • [http://partsregistry.org/Part:BBa_K538204 BBa_K538204]: pLacI + RBS + CspC
  • [http://partsregistry.org/Part:BBa_K538210 BBa_K538210]: pLacI + RBS + Cpn10 + RBS + Cpn60
  • [http://partsregistry.org/Part:BBa_K538211 BBa_K538211]: pLacI + RBS + Cpn60 + RBS + Cpn10
  • [http://partsregistry.org/Part:BBa_K538300 BBa_K538300]: pBAD + RBS + Cpn10
  • [http://partsregistry.org/Part:BBa_K538301 BBa_K538301]: pBAD + RBS + Cpn60
  • [http://partsregistry.org/Part:BBa_K538302 BBa_K538302]: pBAD + RBS + SheDnaK
  • [http://partsregistry.org/Part:BBa_K538303 BBa_K538303]: pBAD + RBS + CspA
  • [http://partsregistry.org/Part:BBa_K538304 BBa_K538304]: pBAD + RBS + CspC
  • [http://partsregistry.org/Part:BBa_K538310 BBa_K538310]: pBAD + RBS + Cpn10 + RBS + Cpn60
  • [http://partsregistry.org/Part:BBa_K538311 BBa_K538311]: pBAD + RBS + Cpn60 + RBS + Cpn10


The four parts containing 2 RBSes and coding regions were designed the way they are, because Cpn10 and Cpn60 should be coexpressed for them to be functional, according to literature. Coexpression can be achieved by transforming E. coli with two plasmids, but we feel this operon-like brick structure is also an elegant solution. [http://partsregistry.org/Part:BBa_K538210 BBa_K538210] should be functionally identical to [http://partsregistry.org/Part:BBa_K538211 BBa_K538211], as should [http://partsregistry.org/Part:BBa_K538310 BBa_K538310] and [http://partsregistry.org/Part:BBa_K538311 BBa_K538311] be. They only differ in the order of their coding regions, which resulted from us planning to assemble the operons by appending [http://partsregistry.org/Part:BBa_K538100 BBa_K538100] to [http://partsregistry.org/Part:BBa_K538201 BBa_K538201] and [http://partsregistry.org/Part:BBa_K538301 BBa_K538301], or by appending [http://partsregistry.org/Part:BBa_K538101 BBa_K538101] to [http://partsregistry.org/Part:BBa_K538200 BBa_K538200] and [http://partsregistry.org/Part:BBa_K538300 BBa_K538300].

Even though we tried many different assembly protocols to attain complete CryoBricks, our progress was impaired by untracable technical difficulties. At the time of this writing, only two protein generators - the Cpn10 and CspC generators under control of pLacI - are assembled and characterised, and because neither of them could be transferred to the pSB1C3 backbone succesfully, none of the CryoBrick generators are available from the registry for the time being.

Fortunately, we were able to characterise the two generators we assembled. Refer to the characterisation page for information on how they were characterised, the results of said characterisation, and our conclusion regarding the parts.

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