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 generate extra cuts during JUMP assembly thus unable to be assembled. | + | Designing mistake: There were two BsaI cutting sites in the sequence which will generate extra cuts during JUMP assembly thus unable to be assembled correctlly. |
==JUMP Assembly== | ==JUMP Assembly== | ||
Revision as of 19:02, 5 October 2022
Pseudomonas fluorescens Metallothionein
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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 correctlly.
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
| 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