Team:NYMU-Taipei/ymic2.html

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                 <li><a title="Methods" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
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Revision as of 20:46, 26 October 2012

NYMU iGEM

Experiment Design

Into the cell
By cloning LLO and invasin into the E.coli, the bacteria can get into the amoeba and live inside it. Invasin helps the cell to be endocytosed by the host. Then Listeriolysin O makes the cell escaped from phagosomes in the cytoplasm of the host.

Cadmium accumulation
To enhanced E.coli’s ability of gathering cadmium ions, we clone smtA and mnt gene into the E.coli. SmtA is a cadmium binding metallothionein, which can bind to cadmium ions. Mnt is an ion transporter that pumps cadmium ions inside the bacteria.


The main idea of how E.coli and Dictyostelium discoideum system works.

 
---smtA---
Metallothionein (MT) is a family of cysteine-rich, low molecular weight (MW ranging from 500 to 14000 Da) proteins. MTs have the capacity to bind both physiological (such as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals through the thiol group of its cysteine residues(1).
Synechococcus PCC. 7942 gene smtA encodes the protein designated to be a metallothionein (MT). While expressing this gene in E.coli, it can increase cadmium ion tolerance of E.coli and so does the accumulation of cadmium ion(2)(3).



---mnt---
Mnt gene is the Mg2+/Cd2+ transporter which belongs to ABC transporters system. The ATP-binding cassette (ABC) transporters form one of the largest known protein families, and are widespread in bacteria, archaea, and eukaryotes. They couple ATP hydrolysis to active transport. The structure of a prokaryotic ABC transporter usually consists of three components; typically two integral membrane proteins each having six transmembrane segments, two peripheral proteins that bind and hydrolyze ATP, and a periplasmic (or lipoprotein) substrate-binding protein. Many of the genes for the three components form operons as in fact observed in many bacterial and archaeal genomes.(4)

Cloning mntA gene into E.coli can increase Cd2+ uptake.




Biosafety Consideration

--- Precaution against Horizontal gene transfer – Living inside of amoeba---
Horizontal gene transfer(HGT) is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent HGT, we must make sure our genetic modified E.coli will never join their wild type relations. Our solution is to put the engineered E.coli into amoeba, so the E.coli cannot contact the environment directly. Whenever wild type bacteria break into the amoeba, they will be digested.


--- Precaution against becoming invasive species - Kill switch of Amoeba
---
To make sure our genetic modified organisms will stay in the area where we want them to clean up cadmium ions, we also build a kill-switch that will switch on when there is no cadmium ion in the soil. The kill-switch has two man-made operons. The first one includes zinTp(previous name pYodA), this promoter activity is induce by cadmium ion; and its constructive gene is lacI. The other one includes BBa_R0010, the promoter whose activity is inhibited by the protein lacI; and the constructive gene DdaifA and HlyA(BBa_K223054). DdaifA is a protein that cause the apoptosis of Dictyostelium discoideum cell. HlyA is a tag that stick after the target protein. With this tag, E.coli will secrete the target protein. When cadmium ions exist, zinTp works and lacI is generated, so the expression of aifA and HlyA is repressed. If there is no cadmium ions, BBa_R0010 will activate the expression of aifA and HlyA, so the protein aifA will be secrete right into the cytoplasm of amoeba, causing amoeba apoptosis.

 
---ZinTp---

ZinTp (pYodA) is a promoter which expresses the downstream gene in the presence of cadmium ion. The activity of this promoter is specific affect by cadmium ion and won’t be induced by other ions like zinc, copper, cobalt, and nickel(5).

Strain AL6 (λФ PyodA–lacZ) was grown aerobically in LB medium at 37°C in presence (●) or absence (○) of cadmium (136±5μM) β-Galactosidase activity in the presence (■) or absence (□) of tested agents was also measured.

 
---DdaifA---

Apoptosis-inducing factor (AIF) is involved in a caspase-independent cell death pathway. Dictyostelium discoideum has a homolog of mammalian AIF, which could cause nuclei breakdown, leading the cell to the apoptosis program(6).

DdAIF induces damage of mammalian and Dictyostelium nuclei in a cell-free system. (E) shows the quantification of damaged nuclei induced by cytoplasmic extracts. Damaged nuclei were assessed by the disappearance of the nucleoli. One hundred nuclei were counted in each condition. Histograms are the mean of three independent experiments. Nu, nucleolus; Dnu, dying cells nucleolus. Dictyostelium cells (control CE), Dictyostelium cells treated with PPIX to cause cell death (dying cells CE). Cytoplasmic extracts of dying cells were also immunodepleted with anti-DdAIF (dying cells CE -AIF) or with nonimmune serum (dying cells CE NIS)