Difference between revisions of "Part:BBa K4023000"

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===Experiments and Methods===
 
===Experiments and Methods===
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The experimental design can be found on our wiki in the [https://2021.igem.org/Team:Washington/Experiments]. 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.  
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The experimental design can be found on our wiki in the [https://2021.igem.org/Team:Washington/Experiments]. Due to constraints 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.
  
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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.
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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.  
  
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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 [https://2019.igem.org/Team:Washington/Experiments 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<sub>D</sub>) can be calculated. This K<sub>D</sub> is a inversely proportional to binding affinity, thus a high K<sub>D</sub> would indicate a low binding affinity, and vice versa.
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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
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===Results===
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===Discussion===
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===References===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 09:53, 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]. Due to constraints 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

Discussion

References

Sequence and Features


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
    COMPATIBLE WITH RFC[1000]