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Biosafety Cadmium Collector

Overview We’ve built the endosymbiotic system to clean up cadmium contamination. The engineered organism can collect cadmium ion in the soil and can be easily gather together; moreover, the device won’t become another pollution source to our environment. Introduction Cadmium contamination is a serious problem in many countries; cadmium can cause respiratory system damage, renal failure, bones softening, and, maybe the most infamous case in Asia, Itai-itai disease. The rice absorbed the cadmium in the soil and the cadmium accumulated in the people eating contaminated rice. The aim of our project is to build up a system that can collect Cadmium ion in the soil and can be easily gather together; besides, we don’t want our device become another pollution source to our environment. We found that the endosymbiotic system between the soil-living amoeba Dictyostelium discoideum and E.coli would be our favorite solution.

Cadmium ions were absorbed by amoeba then accumulated inside of E.coli

Why Dictyostelium discoideum Dictyostelium discoideum is a soil-living amoeba, so it is a good choice to collect cadmium ion in soil. In addition, the amoeba naturally has endosymbiont inside the cell. The E.coli living in amoeba accumulates cadmium ions, just like an organelle, the E.coli acts as a compartment preventing its host from cadmium poisoning. Furthermore, keeping the E.coli inside the amoeba avoid unexpectedly gene exchange between engineered E.coli and other wild types bacteria. Moreover, Dictyostelium discoideum chemotaxis is well-studied and can be easily gather by adding cAMP.

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. ---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 . 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 .

---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.

      mntA structure of Lactobacillus plantarum 

Kill switch of E.coli In case that the E.coli get rid of amoeba, we design a mechanism to cause the death of bacteria. Endolysin is an enzyme that degrades the bacterial peptidoglycan cell wall, resulting in lysis of the bacterial cell. Holins are small membrane proteins that let endolysin get to where it works. Anti-holin acts as anti-toxin by binding to holin, inhibiting it activity. LuxR promoter is a promoter which will only express downstream gene when the protein LuxR and signal molecule AHL, which is produced by protein LuxI, are both exist inside the bacteria. Engineering continual expression endolysin, holin and LuxR; and LuxR promoter as the promoter for anti-holin gene in E.coli, the bacteria will not survive without AHL. We clone the LuxI gene to the plasmid in Dictyostelium discoideum, making the amoeba to produce AHL. By this way, when E.coli stay in the amoeba, the AHL will be sensed by LuxR, maintaining the transcription of anti-holin gene, preventing the E.coli from lysing. But when the E.coli leave the amoeba, there will be no AHL so no anti-holin will be produced and the E.coli will be kill by endolysin.

Kill switch of E.coli 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 .

---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 .

Methods and Materials

Kill-switch of amoeba Cloning zinTp into psb1c3 plasmid. We got the zinTp sequence from RegulonDB (http://regulondb.ccg.unam.mx/operon?term=ECK120034090&format=jsp). Amplification was performed for 40 cycles of denaturation for 30 sec at 94 °C, annealing for 1 min at 59 °C and polymerization for 1 min at 68 °C. A PCR product containing the zinTp was cloned into plasmid psb1c3. The plasmid we used was got from iGEM 2012 distribution kit. The resulting plasmid was transformed into E. coli DH5αcompetent cells. Characterizing of zinTp. We ligated the zinTp with GFP generator(BBa_E0840) and tested the function of zinTp. The transgenic E.coli contains zinTp+GFP, which should fluoresce when Cd2+ exist. We added different concentration of cadmium ion into LB medium and compared the fluorescence level of E.coli which contain zinTp+GFP, pTetR+GFP (BBa_I13522), and psb1c3 backbone only(wildtype control).

We grew E.coli to OD595=0.3~0.4, then added cadmium ion into micro centrifuge tubes. The E.coli was incubated at 37°C. We tested the fluorescence level of GFP. The excitation wavelength is 501nm and the emission wavelength is 511nm. The timeline and cadmium ion concentration was shown below.

    time

cadmium concentration 0 min 15 min 30 min 1 hr 2 hr 4 hr 8 hr 0 μM 20 μM 50 μM 100 μM 200 μM

Enhancing absorption and Tolerance to cadmium ions Cloning mnt into psb1c3 plasmid. We got the mnt sequence from NCBI (http://www.ncbi.nlm.nih.gov/gene/954579 ; http://www.ncbi.nlm.nih.gov/gene/954580 ; http://www.ncbi.nlm.nih.gov/gene/954581 ). Next, we grew Synechocystis sp. PCC 6803 and cloned the mnt gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells. The plasmid we used was got from iGEM 2012 distribution kit. Cloning smtA into psb1c3 plasmid. Firstly, we got the smtA sequence from NCBI (http://www.ncbi.nlm.nih.gov/gene?term=SmtA%20Synechococcus%20elongatus%20PCC%207942). Next, we grew Synechococcus elongatus PCC 7942 and cloned the smtA gene into psb1c3. Then we transformed the plasmid into E. coli DH5αcompetent cells.

Characterizing of smtA. We cloned the smtA gene into the plasmid Ptrc-PNSII-Strep. This plasmid includes a promoter Ptrc. We compared the tolerance level between Ptrc-smtA-PNSII-Strep E.coli and E.coli that did not contain smtA(Ptrc-PNSII-Strep plasmid as a wild type control). We tested the growth curve of E.coli with different cadmium concentration.

The E.coli was grown to OD595=0.3~0.4, then we added 200λ broth into 6.cc liquid LB medium with Streptomycin 25 μg/ml. We incubated the E.coli at 37 °C. The timeline and cadmium ion concentration was shown below.

    time

cadmium concentration 0 hr 1 hr 2 hr 4 hr 8 hr 16hr 0 μM 50 μM 100 μM 200 μM 500 μM

Results and Discussion Cloning zinTp into psb1c3 plasmid.

We got the right plasmid which contained zinTp. Characterizing of zinTp.

In this fig we could see that before 60 min, the fluorescence level of zinTp+GFP steadily increased, while BBa_I13522 (positive control) remained stable around 35000 au as time gone by. At 60 min, zinTp reached its maximum transcription rate so the fluorescence level stop increasing around 55000 au.

      	   Fluorescence level at 15mins after Cd2+  added 

This fig shows the fluorescence level of different cadmium concentration. We can see that BBa_I13522 remained stable from different cadmium concentration, while the fluorescence level of zinTp+GFP increased as the cadmium concentration increased. We could also tell that the transcription rate of zinTp is fairly low when there is no cadmium ion.

Cloning mnt into psb1c3 plasmid.

Mnt gene was successfully cloned into pSB1C3 plasmid.

Cloning smtA into psb1c3 plasmid.

smtA gene was successfully cloned into pSB1C3 plasmid.

Characterizing of smtA.

             Cadmium 0/200/500μM Medium 

This fig shows that smtA enhances the tolerance level to cadmium ion. In wild type E.coli, OD595 differ from between 0/200/500μM cadmium concentration. E.coli hardly grow in 500μM cadmium medium. However, the E.coli which can express smtA seem to show no significant different between 0 & 200μM cadmium medium.

8 hours after cadmium added OD of WT E.coli decrease when cadmium concentration increase. The E.coli which can express smtA seem to have stronger resistance to cadmium toxicity and the E.coli concentration show no significant different from 0 to 200μM cadmium medium.

Obviously, the E.coli which could express smtA growth better than wild type in 200μM cadmium medium. Conclusion and References conclusion 1. We have successfully cloned zinTp into pSB1C3 2. We have successfully cloned zinTp-GFP into pSB1C3 3. We have characterized zinTp function. The expression of downstream gene will increase when added cadmium ion into LB medium, and the maximum transcription rate occurs around 60 min after added 200μM cadmium. 4. We have successfully cloned mnt into pSB1C3 5. We have successfully cloned smtA into pSB1C3 6. We have characterized smtA function. When expressing smtA, it enhances E.coli tolerance to cadmium. References 1. Sigel, A.; Sigel, H.; Sigel, R.K.O., ed. (2009). Metallothioneins and Related Chelators. Metal Ions in Life Sciences. Cambridge: RSC Publishing 2. .Jianguo Shi, William P. Lindsay, James W. Huckle, Andrew P. Morby and Nigel J. Robinson. Cyanobacterial metallothionein gene expressed in Escherichia coli Metal-binding properties of the expressed protein. FEB.5 11081 Volume 303, number 2,3, 159-163 3. Sode et al. (1998) Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene. J Mar Biotechnol 4. ZHIQI HAO,1 SHAOLIN CHEN,1 AND DAVID B. WILSON1,2, Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus plantarum, APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Nov. 1999, p.4746–4752 Vol. 65, No. 11 5. A. Paskarova, P. Ferianc, J. Kormanec, D. Homerova, A. Farewell, T. Nystrom. Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli, Microbiology (2002), 148, 3801–3811 6. Damien Arnoult, Ire`ne Tatischeff, Je´rome Estaquier, Mathilde Girard, Franck Sureau, Jean Pierre Tissier, Alain Grodet, Marc Dellinger, Francois Traincard,Axel Kahn,Jean-Claude Ameisen, and Patrice Xavier Petit. “On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor during Dictyostelium discoideum Cell Death”, Molecular Biology of the Cell Vol. 12, 3016–3030, October 2001