Difference between revisions of "Part:BBa K187000"
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pAB is one of two universal plasmids for the BioBytes standardized in vitro assembly method. The other universal plasmid, pBA, is entered as <partinfo>BBa_K187001</partinfo>. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends.   
 
pAB is one of two universal plasmids for the BioBytes standardized in vitro assembly method. The other universal plasmid, pBA, is entered as <partinfo>BBa_K187001</partinfo>. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends.   
  
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In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB.  This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette.[[pAB "Image:https://static.igem.org/mediawiki/2009/9/95/PAB.png"]]]]
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In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB.  This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette. Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part (see parts <partinfo>BBa_K187365</partinfo> and <partinfo>BBa_K187366</partinfo>). After amplification, treatment with USER mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB Byte.
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Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part. To create an AB Byte from pAB, the universal primers pAB_F and pAB_R are usedTo prepare parts inserted in pAB for assembly, primers <partinfo>BBa_K187365</partinfo> and <partinfo>BBa_K187366</partinfo> should be used.
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The BioBytes system is based on a bead platform where constructs with long sticky ends are annealed together sequentially. This system is designed to enable the single day assembly of plasmids composed of up to about half a dozen premade parts. Complementary to existing BioBrick standards for DNA assembly, we expect this system to be useful for rapid generation of large constructs wherever an interpart spacing of 37 bp can be tolerated.
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From this point the Bytes are ready to be used in the bead platform construct. For more information please see the BioBytes Standard RFC #47 and [http://2009.igem.org/Team:Alberta/Project/ByteCreation].
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Using the plasmids pAB and pBA, as well as universal primer sequences for the PCR extraction of bricks from the plasmids, anyone will be able to use the BioBytes system for their own rapid assembly. The design ensures that the existing catalog of BioBrick parts is compatible with the system, and can be easily cloned into the universal plasmids for brick generation. We believe that this new system is ready for both use and formalization, and we have prepared a standard ([http://bbf.openwetware.org/RFC.html#BBF_RFC_47:_BioBytes_Assembly_Standard BBF RFC 47]) detailing the use of BioBytes.
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Revision as of 00:32, 22 October 2009

pAB is one of two universal plasmids for the BioBytes standardized in vitro assembly method. The other universal plasmid, pBA, is entered as BBa_K187001. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends.

In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette. Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part (see parts BBa_K187365 and BBa_K187366). After amplification, treatment with USER mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB Byte.

From this point the Bytes are ready to be used in the bead platform construct. For more information please see the BioBytes Standard RFC #47 and [http://2009.igem.org/Team:Alberta/Project/ByteCreation].