Difference between revisions of "Part:BBa K4390013:Design"
 
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This part has been codon optimised for expression in E. coli K12. The native stop codon has been removed and replaced with a double stop codon.
 
This part has been codon optimised for expression in E. coli K12. The native stop codon has been removed and replaced with a double stop codon.
  
−
'Designing mistake:' There were two BsaI cutting sites in the sequence which will cause extra cuts which will cause the assembly fails.
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Designing mistake: There were two BsaI cutting sites in the sequence which will generate extra cuts during JUMP assembly thus unable to be assembled correctly.
  
 
==JUMP Assembly==
 
==JUMP Assembly==

Latest revision as of 19:02, 5 October 2022


Pseudomonas fluorescens Metallothionein


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 214


Design Notes

Design Notes

This part has been codon optimised for expression in E. coli K12. The native stop codon has been removed and replaced with a double stop codon.

Designing mistake: There were two BsaI cutting sites in the sequence which will generate extra cuts during JUMP assembly thus unable to be assembled correctly.

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

Habjanič, J., Zerbe, O., Freisinger, E., 2018. A histidine-rich Pseudomonas metallothionein with a disordered tail displays higher binding capacity for cadmium than zinc. Metallomics. https://doi.org/10.1039/c8mt00193f

References