Difference between revisions of "Part:BBa K1807000:Design"

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__NOTOC__
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<partinfo>BBa_K1807000 short</partinfo>
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<partinfo>BBa_K1807000 SequenceAndFeatures</partinfo>
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===Source===
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''Escherichia coli''; Registry of Standard Parts
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===Design Notes===
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Above is a diagram of our Adapter BioBrick in the pSB1C3 vector.
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Our design incorporates a SmaI restriction digestion step and subsequent use of the cut Adapter as a vector in a Gibson Assembly Reaction or any other assembly method based on flanking overlapping sequences (as depicted below).
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BBa_K1807000 was designed as a blue-white screening device that would also be easily used in Gibson Assembly.
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Surrounding the lacZ alpha coding sequence are two SmaI restriction sites (CCC/GGG). SmaI is a blunt-end endonuclease- we used it to simulatenously linearize our vector and remove the lacZ alpha coding sequence. Our BioBricks contained overhangs that make them compatible with the SmaI-digested BBa_K1807000.
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The overhang sequences used to make this part are as follows:
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'''BBa_K1807000 Assembly''' 5'end Overhang (complementary to pSB1C3): cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGAG
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'''BBa_K1807000 Assembly''' 3'end Overhang (complementary to pSB1C3): TACTAGTAGCGGCCGCTGCAGtccggcaaaaaagggcaag
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'''The subparts of the device are as follows:'''
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BBa_R0011, 
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BBa_B0034,
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BBa_E0038,
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BBa_B0015.
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== How to Use this Part ==
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The '''Splice-In Flanking Sequences''' (30bp each) that allow any Protein Coding Sequence to be inserted in place of lacZ alpha:
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UPSTREAM of CDS : '''5' AGCACATACGAGAAAGAGGAGAAATACCCC 3'''' + Start Codon (ATG) + CDS
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DOWNSTREAM of CDS: CDS + Stop Codon(s) + '''5' GAGCCTTTCGTTTTATTTGATGCCTGGCCC 3''''
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iGEM York 2015 recommends that teams and researchers use the above-listed '''Splice-In Flanking Sequences''' together with BBa_K1807000 for Gibson Assembly or any other cloning or assembly method relying on complementary overhangs.
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'''''Cloning'''''
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The '''Splice-In Flanking Sequences''' can be incorporated in a pair of primers as 5' overhangs. Such a primer pair can be used to directly amplify a target CDS from genomic DNA extraction, purified plasmid DNA or genomic (plasmid) DNA from a colony (as in a Colony PCR).
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If cloning from an operon or just a protein domain, for efficient translation it is important to include '''Start''' and '''Stop''' Codons. Those can be added to the overhang sequence as depicted above.
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'''''Synthetic Gene Design'''''
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If designing a G Block fragment or any other dsDNA that is to be chemically synthesized the '''Splice-In Flanking Sequences''' can be added to each end.
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===References===

Latest revision as of 18:23, 18 September 2015

Protein generator device suitable for blue-white screening and Gibson Assembly.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

Escherichia coli; Registry of Standard Parts


Design Notes

Above is a diagram of our Adapter BioBrick in the pSB1C3 vector. Our design incorporates a SmaI restriction digestion step and subsequent use of the cut Adapter as a vector in a Gibson Assembly Reaction or any other assembly method based on flanking overlapping sequences (as depicted below).


BBa_K1807000 was designed as a blue-white screening device that would also be easily used in Gibson Assembly. Surrounding the lacZ alpha coding sequence are two SmaI restriction sites (CCC/GGG). SmaI is a blunt-end endonuclease- we used it to simulatenously linearize our vector and remove the lacZ alpha coding sequence. Our BioBricks contained overhangs that make them compatible with the SmaI-digested BBa_K1807000.

The overhang sequences used to make this part are as follows:

BBa_K1807000 Assembly 5'end Overhang (complementary to pSB1C3): cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGAG

BBa_K1807000 Assembly 3'end Overhang (complementary to pSB1C3): TACTAGTAGCGGCCGCTGCAGtccggcaaaaaagggcaag

The subparts of the device are as follows: BBa_R0011, BBa_B0034, BBa_E0038, BBa_B0015.


How to Use this Part

The Splice-In Flanking Sequences (30bp each) that allow any Protein Coding Sequence to be inserted in place of lacZ alpha:

UPSTREAM of CDS : 5' AGCACATACGAGAAAGAGGAGAAATACCCC 3' + Start Codon (ATG) + CDS

DOWNSTREAM of CDS: CDS + Stop Codon(s) + 5' GAGCCTTTCGTTTTATTTGATGCCTGGCCC 3'

iGEM York 2015 recommends that teams and researchers use the above-listed Splice-In Flanking Sequences together with BBa_K1807000 for Gibson Assembly or any other cloning or assembly method relying on complementary overhangs.

Cloning

The Splice-In Flanking Sequences can be incorporated in a pair of primers as 5' overhangs. Such a primer pair can be used to directly amplify a target CDS from genomic DNA extraction, purified plasmid DNA or genomic (plasmid) DNA from a colony (as in a Colony PCR).

If cloning from an operon or just a protein domain, for efficient translation it is important to include Start and Stop Codons. Those can be added to the overhang sequence as depicted above.


Synthetic Gene Design

If designing a G Block fragment or any other dsDNA that is to be chemically synthesized the Splice-In Flanking Sequences can be added to each end.


References