Part:BBa_K4390010
Mytilus galloprovincialis Metallothionein
Usage and Biology
Metallothionein (MT) is a small protein (around 6-7 kDa) which is rich in cysteine. These thiol group in cysteines provide ability to chelate almost all heavy metal ions including Cd2+, Hg2+, Pb2+ and As3+, but had been shown that has higher binding affinity with Hg2+ (Manceau, A. et al., 2019). The ability of chelating heavy metals provides the metal tolerance for its hosts. For its ability to binding heavy metal strongly, this part can be used to build structure which can capture heavy metal ions in aqueous environment. This MT sequence was obtained from Mytilus galloprovincialis, as an isoform which contains higher cysteine frequency with 23 in 72 amino acids (Vergani, L. et al., 2007) and designed as a C part for JUMP assembly (Valenzuela-Ortega M and French C., 2021).
Design
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.
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.
Usage and Results
Mytilus edulis MT sequence was assembled into plasmid BBa_J428341 along with BBa_K4390017 and BBa_K4390016. After transformation, we performed colony PCR to verify the assembly was successful (Figure 1).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |