Part:BBa_K4023000
Modified MTIA
Modified MTIA codes for a metallothionein IA protein modified to possess specificity to arsenite in all its 7 metal binding sites. This part is optimized for expression in E.coli. When expressed, the metallothionein IA protein is intended to sequester arsenite entering E.coli. The part can therefore be used in whole cell remediation of arsenite.
Biology and Usage
Metallothionein are small proteins containing a high percentage of conserved cysteine residues. In general, it has 7 metal binding sites, 3 for zinc and 4 for Cadmium and can bind a variety of different heavy metals, with different binding affinity. Metallothionein-IA is a metallothionein protein found in humans. It is broadly expressed in the liver, which matches its known function of detoxification of heavy metals. It is also known to protect against oxidative stress and carcinogens.
We modified all the 7 binding sites of Metallothionein IA to be more specific to Arsenic. This would potentially increase the concentration of Arsenic accumulated by the protein in a single bacteria cell. Additionally, we also modified the protein backbone to energetically stabilize the protein, particularly when it is bound to Arsenic. As MTIA is a eukaryote gene, the sequence needed to be optimized for expression in E.coli. Hence the gene has been optimized with the IDT codon optimization tool and Benchling, looking out for GC content, uridine content and hairpin loops.
Our initial project goal focused on the accumulation of Arsenic from a human gut environment. This resulted in the use of mammalian proteins. The 4MT2 rat metallothionein protein was used as a template model for the MT1A human metallothionein sequence. Despite the project’s transition into an environmental focus, the mammalian protein modeling serves as a proof of concept for modifications made in metallothionein proteins. Our protein modeling work provides a foundation for future work on plant metallothionein.
Characterization of Metallothionein
Characterization of composite part was performed by wetlab and drylab protein modelling.
Drylab
Through Homology Modeling of MTIA with 4MT2 as template, we successfully modeled the structure of MTIA. With further designing, the modified MTIA is shown below.
Experiments and Methods
The experimental design can be found on our wiki in the Experimentstab. Due to resitrictions imposed by the COVID 19 situation, the gene was synthesized via IDT, and transformation and verification was performed by UW Biofab. We collected the successfully transformed and streaked plates of BL21 DE3 E.coli from UW Biofab and induced protein expression by inoculating a colony of transformed E.coli in MagicMedia™ E. coli Expression Medium overnight. An aliquot of the induced bacteria were lysed for protein expression, while the rest were collected for metal tolerance assay.
Protein concentration of lysate was analyzed with Nanodrop, and protein concentration was diluted to 2mg/ml with 1x Laemmli buffer. The samples were then loaded into precast SDS gel and ran for ~30min at 200V. The gel was then stained with Coomassie Blue for 2 hours and destained with destaining solution, changed every 30min accompanied by gentle agitation.
The metal tolerance assay involves the determination of the minimum inhibitory concentration of Arsenite on the successfully transformed bacteria. Briefly, initial concentration of bacteria was determined and the induced bacteria were diluted. Meanwhile LB broth containing various concentration of Sodium Arsenite solution (between 0mM to 10mM) was prepared. Subsequently, 25ul of diluted induced bacteria and 175ul of LB broth with Sodium Arsenite were added in 96 well microplates. The plates were incubated at 37 degree Celsius, and the absorbance at OD600 was taken after 20hrs of incubation.
Results
Due to the limited amount of time we had in the lab, the data we gathered are preliminary and requires further experimentations to improve reliability and accuracy. Nevertheless they are promising data and bodes well to the feasibility of our system design and of the intended function of the modified protein.
SDS PAGE
The size of MT proteins are around 6kDa. From the gel, MTs are found in all 6 lanes, indicating that the 6 cultures of bacteria containing 6 different plasmids all managed to express MTs successfully.
Metal Tolerance
E.coli containing MTIA, 4MT2 and Modified MTIA (UWMT1) all experienced a decrease in viability as concentration of arsenite increased. There is no significant difference between the data points at each concentration of Arsenic, hence the arsenite tolerance of E.coli containing the 3 plasmids are likely similar. This suggests that the Modified MTIA (UWMT1) will not increase E.coli susceptibility to arsenite and thus is feasible for use in whole cell remediation of arsenite.
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