Difference between revisions of "Part:BBa K4023000"

Revision as of 09:33, 21 October 2021

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. 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

Dry lab

Experiments and Methods

The experimental design can be found on our wiki in the [1]. Briefly, the due to constraints imposed by the COVID 19 situation, gene was synthesized via IDT, and transformation and verification was performed by Biofab UW. We collected the successfully transformed and streaked plates of BL21 DE3 E.coli from 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.

The protein was expressed in WK6 overnight and released from the periplasm of the bacteria using a TES buffer solution. The periplasm extract was then run through a nickel his-trap column for protein purification. After protein purification, the protein was biotinylated and the protein concentration was quantified using a spectrophotometer.

The binding affinity of the mutant and wild-type binder was then quantified using biolayer interferometry (BLI). The specific machine we used was the OCTET Red96 plate reader, using Super Streptavidin Sensors. This procedure, outlined in our experiments page, measures the association and dissociation rates for the binder to CBD at different CBD concentrations. Using these two values, the equilibrium dissociation constant (K_D) can be calculated. This K_D is a inversely proportional to binding affinity, thus a high K_D would indicate a low binding affinity, and vice versa. Sequence and Features