Difference between revisions of "Part:BBa K4390022:Design"

 
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===Design Notes===
 
===Design Notes===
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This compound (level 1) part was assembled using JUMP, a type IIS assembly method, so all basic (level 0) parts had to have the correct JUMP fusion sites flanking, as well as BsaI sites for level 1 assembly and BsmBI sites for level 0 assembly.
 
  
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==Design Notes==
  
 +
==JUMP Assembly==
  
 +
This part was designed with JUMP assembly (a Type IIS assembly method) in mind. All basic parts were designed to be ordered with flanking BsaI and BsmBI sites, as well as the JUMP fusion sites. When basic parts were being ordered in, they would follow the general structure of
  
 +
BsmBI recognition site :: BsaI recognition site :: JUMP 5’ fusion site :: Part sequence :: JUMP 3’ fusion site :: BsaI recognition site :: BsmBI recognition site
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 +
OR
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CGTCTCGGTCTCC [JUMP 5’ fusion site] :: Part sequence :: [JUMP 3’ fusion site] :: CGAGACCTGAGACG
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{| class="wikitable" style="margin:auto"
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|+ JUMP fusion sites
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|-
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! Part type !! 5’ Fusion site !! 3’ Fusion site
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|-
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| P (Promoter) || GGAG || TACT
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|-
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| R (Ribosome Binding Site) || TACT || AATG
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|-
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| N (N-terminus) || AATG || AGCC
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|-
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| O (Open Reading Frame)|| AGCC || TTCG
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|-
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| C (C-terminus)|| TTCG || GCTT
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|-
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| T (Terminator)|| GGCT || CGCT
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|}
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 +
 +
Composite parts that were ordered in synthetically were designed and ordered with the correct fusion sites, as if they had been assembled from basic parts.
 +
 +
For basic parts in the coding sequence (N, O and C), some extra design considerations took place. Parts that did not end with a C part (everything except C, OC and NOC parts) had all stop codons removed, and nucleotides were either inserted or deleted so that the fusion site would not produce a scar. The overall rule for alignment is that the next codon starts immediately after the fusion site. This means that parts begin with the ATG in the R-N fusion site (AATG), the GCC in the N-O fusion site (AGCC) produces an alanine as a scar and the TCG in the O-C (TTCG) fusion site produces serine as a scar. What we often did was remove some nucleotides or codons, and then the scar would regenerate nucleotides or codons that were there before, so less amino acids would be inserted in the composite product.
  
 
===Source===
 
===Source===

Latest revision as of 18:40, 4 September 2022


Saccharomyces cerevisiae MT expression construct


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 506
    Illegal PstI site found at 584
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
    Illegal PstI site found at 506
    Illegal PstI site found at 584
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 506
    Illegal PstI site found at 584
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 506
    Illegal PstI site found at 584
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Design Notes

JUMP Assembly

This part was designed with JUMP assembly (a Type IIS assembly method) in mind. All basic parts were designed to be ordered with flanking BsaI and BsmBI sites, as well as the JUMP fusion sites. When basic parts were being ordered in, they would follow the general structure of

BsmBI recognition site :: BsaI recognition site :: JUMP 5’ fusion site :: Part sequence :: JUMP 3’ fusion site :: BsaI recognition site :: BsmBI recognition site

OR

CGTCTCGGTCTCC [JUMP 5’ fusion site] :: Part sequence :: [JUMP 3’ fusion site] :: CGAGACCTGAGACG

JUMP fusion sites
Part type 5’ Fusion site 3’ Fusion site
P (Promoter) GGAG TACT
R (Ribosome Binding Site) TACT AATG
N (N-terminus) AATG AGCC
O (Open Reading Frame) AGCC TTCG
C (C-terminus) TTCG GCTT
T (Terminator) GGCT CGCT


Composite parts that were ordered in synthetically were designed and ordered with the correct fusion sites, as if they had been assembled from basic parts.

For basic parts in the coding sequence (N, O and C), some extra design considerations took place. Parts that did not end with a C part (everything except C, OC and NOC parts) had all stop codons removed, and nucleotides were either inserted or deleted so that the fusion site would not produce a scar. The overall rule for alignment is that the next codon starts immediately after the fusion site. This means that parts begin with the ATG in the R-N fusion site (AATG), the GCC in the N-O fusion site (AGCC) produces an alanine as a scar and the TCG in the O-C (TTCG) fusion site produces serine as a scar. What we often did was remove some nucleotides or codons, and then the scar would regenerate nucleotides or codons that were there before, so less amino acids would be inserted in the composite product.

Source

Sayers, Z., Brouillon, P., Svergun, D.I., Zielenkiewicz, P., Koch, M.H.J., 1999. Biochemical and structural characterization of recombinant copper-metallothionein from Saccharomyces cerevisiae. Eur J Biochem. https://doi.org/10.1046/j.1432-1327.1999.00451.x


References