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Abstract

Cadmium is the heavy metal which is very harmful to a creature.　The pollution of cadmium brings a serious problem for natural environments. It is very important to collect this efficiently. We tried to solve this problem with recombination Escherichia coli which our team made in this year. We decided to make two kinds of recombination Escherichia coli. One traps cadmium on the outer membrane. It produces fusion protein with the heavy metal binding protein and outer membrane protein and it traps cadmium. Another collects other Escherichia coli. It produces aspartic acid of positive chemotaxis factor to Escherichia coli.

Is it necessary to distribute it to two kinds of why? We suggest the following theory for this question.

"One E. coli has One function theory"

There is the possibility that the following demerit occurs when I summarize two functions in one.The first is that the ability of the Escherichia coli situation decreases by producing multiple protein at a time.The second is that subsequent editing becomes difficult when we complete a gene cassette once. The merit in case of one kind is next. At first it is that a function becomes very simple.We can make Escherichia coli which was specialized in it by a function becoming simple. The number of Escherichia coli just leads to the level of the function, analysis becomes easy.

Our Project

We make various systems by cell-cell communication. We keep one function in one E. coli. This means to make simple plasmid. The following is one example. We’d like to collect cadmium in water. Therefore we use two kinds of E.coli. One catches Cadmium. The other attracts all E.coli by using chemoattractant. Catches E.coli displays metallothionein a protein combines a heavy metal. Cadmium is a kind of heavy metal. The other synthesizes aspartic acid (Asp) one kind of chemoattractant. All E.coli gather in the E. coli synthesizes Asp. To use these E.coli, finally cadmium will be caught.

When we collect cadmium in the water, if use only catching E. coli(Blue),they go away from cadmium. Because they have negative chemotaxisin contrast with cadmium. Consequently, they don't catch cadmium very much.[fig1-A] In constract, if we use two kind of recombinant Escherichia coli(E. coli);gathering E. coli(Red) and catching E. coli(Blue), we can build the creature system which collects cadmium while sitting at cadmium by using two kinds of Escherichia coli.[fig1-B] It is this theoretical merit that we can suggest two kinds of possibility that meeting condition and collection quantity of Escherichia coli are changed by changing the ratio of Escherichia coli in addition to cadmium solution.

We constracted plasmid that synthesize aspartic acid in the presence of cadmium. Part's namber are BBa_K896008(promoter),(RBS),BBa_C0083(Protein coding sequences),(Double terminator). BBa_K896008(zinTp) is a promoter inducing gene expression only in existence of cadmium. AspA mainly catalyzes the deamination of L-aspartic acid to produce fumarate and ammonium ion. As a result of having cultured it in the nutrient medium including the substrate, we confirmed synthesis of aspartic acid by thin layer chromatography.

We are constracting this plasmid now. Recombination Escherichia coli having this plasmid becomes the thing with the function to trap cadmium on outer membrane if construction of this plasmid is completed. It is fusion protein with metallothionine(MT);SmtA and adventitia protein;Ag43. The genetic origin of SmtA is Synechococcus PCC. 7942.

Modeling

We consigned modeling of our project to UT-Tokyo.They readily compiled with our requests. Their model is ideal.We really appreciate their jobs We wish a friendly relationship with UT-Tokyo.Thank you so much!! The detail of their jobs are lists below!!

Fig.1
This is random walk of Escherichia coli on "swarming plate".The vertical axis means arbitrary unit.The horizontal axis means to spatial axis. We culture E.coli on "swarming plate". "swarming plate", a solid culture plate containing lower concentration of agar, on that plate, E. coli can swim.

Fig.2
This is positive chemotaxis by E.coli.Assuming that spoted aspartic acid center of "swarming plate". E.coli has positivechemotaxis for aspartic acid. E.coli gather to spoted point.

Fig.3
This is negativechemotaxis by E.coli.Assuming that spoted cadmium center of "swarming plate". E.coli has negaitivechemotaxis for cadomium. E.coli run away from spoted point.

Fig.4
This model describes our project. Assuming cadmium was spotted on center of swarming plate (spatial axis 500). E.coli run away from spotted point (spatial axis 350~450 and 550~650). Then cadmium promoter of transgenic　E.coli operates. So aspartase produced (spatial axis 350~450 and 550~650).

Meｍberｓ of UT-Tokyo

Method

Medium

Trypton broth

Trypton 10g/L

NaCl 10g/L

H2O 1L

Wash medium

Potassium phosphate buffer (pH 7.0), 10⁻²M

MgSO₄, 10⁻³M

potassium ethylenediaminetetraacetate (EDTA), 10⁻⁴M

Chemotaxis medium

Potassium phosphate buffer (pH 7.0), 10⁻²M

potassium EDTA, 10⁻⁴M

L-methionine 10⁻⁶M

Synthesis medium

Sodium hydrogen fumarate 46g/L

Ammonium chloride 17.8g/L

Magnesium sulfate 7 hydrate 0.25g/L

H₂O 1L

Ph8.5with sodium hydrogen

LB medium

Tryptone 10g/L

Yeast extract 5g/L

NaCl 10g/L

(agar 15g/L)

H₂O 1L

2×YT medium

Tripton 16g/L

Yeast extract 10g/L

NaCl 5g/L

(agar 15g/L)

H₂O 1L

M9 swarming agar

M9 salt

1.25 % (v/v) glycerol

0.3 % (w/v) agar

0.1 % (w/v) CaCl₂ solution (after autoclave)

0.1 % (w/v) MgSO₄ solution (after autoclave)

0.03 % (w/v) thiamine (after autoclave)

5 × M9 stock solution

6.4 % (w/v) Na₂HPO₄

1.5 % (w/v) KH₂PO₄

0.25 % (w/v) NaCl

0.5 % (w/v) NH₄Cl

Aspartate synthesis

E.coli K12 transformed with CdP-R.B.S-AspA-d.Ter (BBa_K1342001) previous cultured with cadmium in LB medium (250μM) in 37℃ for 12hr at 120rpm. Adjust Cell mass (OD1.0) and therefor centrifuged 4000rpm for 20 min. Cell pellets ware activated in synthesis medium in 37℃ for 2hr at 120rpm/min.

SDS PAGE

・E. coli JM109 containing CdP-R.B.S-AspA-d.Ter (BBa_K1342001) or normal E. coli was precultured at 37 ℃ for 24 hr with shaking.

・The culture (OD600 0.6-1.0) was used for induction of protein expression by isopropylthio-β-D-galactoside 1mM and Cd2+ 100µM soln. 37℃、o hr, 0.5 hr, 2hr, 6 hr, 24hr.

・200 μl of the induced culture was centrifuge at 13,000 rpm for 1 min at 4 ℃.

・The supernatant was discarded and the pellet was stored at -20 ℃.

・The stored pellet was resuspended in 1 x sample buffer (100 μL), followed by heating at 98 °C for 5 min.

・The lysate was centrifuged at 13,000 rpm for 10 min. at 4℃.

・The supernatant was collected into a new microcentrifuge tube.

・20 μL of the supernatant was analyzed by SDS-PAGE.

TLC assay

We analyzed L-aspartate by thin-layer chromatography (TLC). The equal volume of the supernatant of synthesis medium was added with 7 mg/mL 5-Dimethylamino naphthalene-1-sulfonyl Chloride (dissolved in acetone) and was incubated for more than 30 min at room temperature. Two microliters of the reactant was spotted on a TLC silica plate, and was developed in a mixture of ethyl acetate, pyridine, water, and acetic acid(162：21：11：6 v/v). It is observed by UV irradiation (365nm).

Chemotaxis Assay

Introduction

Chemotaxis of E. coli against aspartic acis was assayed by two methods. One was "swarming assay", and the other was capillary assay. In "swarming assay", we have used "sawarming plate", a solid culture plate containing lower concentration of agar, on that plate, E. coli can swim. We have used capillary containing aspartic acid. The capillary was set in chemotaxis medium containing motile E. coli. The number of E. coli attracted to aspartic acid in capillary was determined by colony formation on LB plate.

Swarming Assay

We assayed chemotaxis of E. coli against cadmium and aspartic acid on soft agar, containing M9 synthetic medium, on that plate E. coli can swim.

Protocol

1.E. coli JM109 was cultured at 30℃ for 12 hours with shaking (50 rpm).

2. Aliquot of the culture was spotted on the center of agar plate.

3. 10 mM L-aspartic acid (40 μl) or 100 mM cadmium chloride (4 μl) was spotted on 25 mm distant from the center of the agar plate.

4. The plate was standed for 5 min at RT.

5. The plate was incubated at 30℃.

Capillary Assay

Object

Assay the chemotaxis of E. coli against aspartic acid with capillary.

Plotocol

1. Preculture E.　coli in tripton broth at 30 ℃ for 12 hr with shaking (50 rpm).

2. Check the motility of E. coli by microscope (× 800).

3. Dilute the E. coli culture (200 μL) with tripton broth (20 mL).

4. Incubate at 30 ℃ with shaking (50 rpm) until log phase (OD600 = ～ 0.2).

5. Aliquote (500 μL) of the culture was centrifuged at 25℃ for 10 min at 3400 x G.

6. Dicard the supernatant.

7. Resuspend the pellets gently with wash medium (50 μL).

8. Repeat 5~7 step.

9. Resuspend the pellets gently with chemotaxis medium (500 μL).

10. Check the motility of E. coli by microscope (× 800).

11. Aliquote (100 μL) of E. coli suspended in chemotaxis medium was set into chamber apparatus.

12. Prepare aspartic acid capillary and negative control capillary that contain 1 μL of chemotaxis medium with and without 10 mM aspartic acid, respectively.

13. Both capillaries (asparate capillary and negative control capillary) were set into chamber apparatus.

14. Incubate at 30 ℃ for 90 mim.

15. The chemotaxis medium in capillary was collected in 100 μL of fresh chemotaxis medium.

16. All chemotaxis medium containing attracted E. coli was plated on LB plate.

17. Incubate at 37 ℃ for 12 hr.

18. Count the number of colony.

Result & Discussion

TLC Assay

Analysis of aspartic acid synthesized by E. coli JM109/[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342001 BBa_K1342001] by thin-layer chromatography (TLC) Lane: 1, 2 no addition into culture (diluted 1:50 and 1:100 by synthesis medium, respectively) 3, additon of cadmium ion (250μM) 4, 5, 6 (diluted 1:50, 1:100 and 1:200 by synthesis medium, respectively)

fig.2
Estimation of aspartic acid content in spot Each intensity of spots indicating the content of aspartic acid, was estimated by Image J. .

SDS PAGE

Analyzed aspartase by E. coli JM109 and E. coli JM109/[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1342001 BBa_K1342001] by SDS-PAGE ,37℃ for 0,6 hr Lane:1,10 Protein Marker(Precision Plus Protein Standards (BIO-RAD)) Lane:2,3 E. coli JM109 Lane:4,5 E. coli JM109/BBa_K1342001 Lane:6,7 E. coli JM109/BBa_K1342001 (Additon of cadmium ion 100μM) Lane:8,9 E. coli JM109/BBa_K1342001 (Additon of cadmium ion 100μM and IPTG 1mM) respectively. OD600=0.74 CdCl2 100μM/IPTG 1mM AspA has 1482bp. It is about 54kDa.

Aspartase synthesize was not detect by CBB stain.

Swarming Assay

Fig.1 E. coli swarming to aspartic acid
L: aspartic acid (10 mM, 40 μl) R: ddH₂O (40 μl)
Incubation: 108 hours
temperature: 30 ℃
E. coli cultured was spotted on the center of agar plate, and chemotaxis was spotted on 25 mm distant from the center.

The swarming circle of aspartic acid (L) was larger than that of ddH₂O (R), and the swarming area around aspartate acid (L) was more larger than that of ddH₂O (R), suggesting that E. coli swarming to aspartate acid.
This experiment indicate that E. coli may have positive chemotaxis for aspartic acid.

Fig.2 E. coli swarming to cadmium
L: cadmium(100 mM, 4 μl) R: ddH₂O (40 μl)
Incubation: 110 hours
temperature: 30 ℃
E. coli cultured was spotted on the center of agar plate, and chemotaxis was spotted on 25 mm distant from the center.

The swarming circle of cadmium (L) was smaller than that of ddH₂O (R), and the swarming area around cadmium (L) was more smaller than that of ddH₂O (R), suggesting that E. coli hate cadmium.
This experiment indicate that E. coli may have negative chemotaxis for cadmium.

Capillary Assay

Result

Fig.1 Left : Aspartic acid, Right : control
There were 13 colonies on Asp plate. One other plate has 1326 colonies.

Discussion

At this method. We didn't assay aspartic acid cehmotaxis of E.coli. We did eleven assays.In this assay,　E.coli formation will appear on aspartic acid plate more than conrol plate. But at result of Fig.1, E.coli colonies appeared control plate more than aspartic acid plate. And all result were not authenticity. There are some problems. One is heat of bacteria spreader. At plating moments. I may burn 'E.coli to death. Next is diameter of capillary. I made capillary by myself. There were various sizes of capillary. I used their capillary. So colonies formation were ununiformity. Last is the number of times of assays. In our source journal (Reference No.7), they assayed more then us.

Future work

We performed a project to collect cadmium using chemotaxis of Escherichia coli this year. But the second plasmid is still building it. We want to perform an experiment such as fig.1-B with two kinds of Escherichia coli as soon as construction is completed. In addition, the chemotactic instruction and control that we used have the applied nature to other projects very much. We want to create the new project using chemotaxis of Escherichia coli.

Reference

[1][http://www.ncbi.nlm.nih.gov/pubmed/12480884 Ferianc, P, et al, Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli, Microbiology, vol.148, 3801–3811, (2002)]
[2]iGEM Goettingen, Homing Coli, https://2012.igem.org/Team:Goettingen/Notebook/Results, (2012)
[3][http://www.ccbi.cam.ac.uk/iGEM2006/index.php/Protocols#Autoclaving_stock_solutions_or_glassware_.28sterilisation.29 iGEM University Of Cambridge, Making swimming Agar, http://www.ccbi.cam.ac.uk/iGEM2006/index.php/Protocols#Autoclaving_stock_solutions_or_glassware_.28sterilisation.29, (2006)]
[4]iGEM UT-Tokyo, L-aspartate chemotaxis assay, https://2011.igem.org/Team:UT-Tokyo/Data/Method, (2011)
[5][http://ecocyc.org/ Keseler et al, EcoCyc, http://ecocyc.org/, (2013)]
[6][http://www.ncbi.nlm.nih.gov/pubmed/17650676 Chmiel, A, Selection and activation of Escherichia coli strains for L-aspartic acid biosynthesis, Polish J. Microbiol, Vol.56, 71-76, (2007)]
[7][http://www.ncbi.nlm.nih.gov/pubmed/4632978 ADLER, J, A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli, Polish J. General Microbiol, Vol.74, 77-91, (1973)]
[8][http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106306/ Sternberg, C, et al, New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria, Polish J. Microbiol, Vol.64, 2240–2246, (1998)]
[9]iGEM UPO-Sevilla, Bacterial crowding, https://2010.igem.org/Team:UPO-Sevilla/Project/Assays, (2010)