From 2014.igem.org

Revision as of 04:07, 16 October 2014

HOME PROJECT PARTS TEAM H.P. SAFETY

Our Project

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
means random walk of E.coli on swarming plate.The vertical axis means arbitrary unit.The horizontal axis means spatial axis.

Fig.2
means positive chemotaxis by E.coli.Assuming that distill aspartate center of swarming plate. E.coli gather to distilled point.

Fig.3
means negativeChemotaxis by E.coli.Assuming that distill cadmium center of swarming plate. E.coli run away from distilled point.

Fig.4
means our project. Assuming that distill cadmium (spatial axis 500). E.coli run away from distilled point (spatial axis 350~450 and 550~650). At that time E.coli`s cadmium promoter is operate. Asparatate is produced by E.coli'

Thank you very much!!

Method

Bold text

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

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

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

・Preculture E.coli holding a plasmid containing a target gene or nomal E.coli.
・Measure OD600 0.6-1.0
・the expression of fusion protein by isopropylthio-β-D-galactoside (IPTG)　and Cd2+ soln.
・Transfer a sample a 200 µl in a microcentrifuge tube
・Centrifuge at 13,000rpm for a minute at 4℃
・Discard supernatant quantitative
・Store pellet at -20 °C
・Thaw pellet and resupend in Sample Buffer (100 µL 1xSample Buffer per samples)
・Heat for 5 minutes at 98 °C
・Centrifuge at 13,000rpm for 10 minutes at 4℃
・Transfer supernatant to a new microcentrifuge tube
・Analyze samples by SDS-PAGE.(Use 20 µL per samples)

TLC assay

We analyzed L-aspartate by thin-layer chromatography (TLC). The same amount added Supernatant of synthesis medium to 7mg/mL 5-Dimethylamino naphthalene-1-sulfonyl Chloride (in Acetone) and incubated more than 30min. Two microliters of the sample was placed onto a TLC silica plate, and developed in a mixture of Ethyl acetate, pyridine, water, and acetic acid(162：21：11：6 V/V).

Chemotaxis Assay

Introduction

Chemotaxis by E.coli was assay by some methods. We assay the chemotaxis of E.coli against aspartate using two methods. One was swarming assay. We have used sawaming plate. swarming plate is medium that contained a low concentration by agar. E.coli can swim to aspartate on the swarming plate. The other was capillary assay. We have used capillary that including aspartate. We set capillary in chemotaxis medium including motile E.coli We have determined the number of E.coli attracted to capillary by colony formation on LB plate.

Swarmming Assay

Capillary Assay

Object
To assay chemotaxis by E.coli against aspartarte using capillary.
Plotocol
1. Preculture E.coli with tripton broth at 30℃ 12 hr 50 rpm
2. Check motility under the microscope(×800).
3. Diluting the E.coli culture 100 times. (tripton broth:E.coli culture＝20mL : 200μL)
4. Incubate 50rpm 30℃ until log phase (OD600 = ～ 0.2)
5. 500㎕ into micro tubes.
6. Centrifuge (25℃ 10min in 3400G)
7. Dicand the supernatant.
8. Add wash medium(50μL)
9. Resuspending pellets gently.
10. Do 5~9 step repeat.
11. Add chemotaxis medium(500μL)
12. Resuspending pellets gently.
13. Check motility under the microscope(×800).
14. E.coli chemotaxis medium into chamber apparatus.(100μL)
15. Prepare the positive and negative control capillary.（1μL of 10mM aspartate chemotaxis medium and chemotaxis medium into capillary）
16. Two capillary into chamber preparation.
17. 90mim 30℃ incubation.
18. Dilute the chemotaxis buffer in capillary`s liquid 100 times.
19. Prating to LBplate.
20. 37℃ 12h incubation.
21. Counting the colony.

Result & Discussion

TLC Assay

Analysis of aspartate from strain E.coli K-12/BBa_K1342001 by thin-layer chromatography (TLC) Lane: 1, not addition cadmium to previous culture of synthesis medium (diluted 1:50 by synthesis medium) 2, (diluted 1:100 by synthesis medium) 3, add cadmium (250μM) 4, (diluted 1:50 by synthesis medium) 5, (diluted 1:100 by synthesis medium) 6, (diluted 1:200 by synthesis medium)

SDS PAGE

1. marker 2. 0hr 3. 0.5hr 4. 2hr 5. 6hr 6. 24hr 7. Marker marker:Precision Plus Protein Standards (BIO-RAD)

OD600=0.74 CdCl2 100μM/IPTG 1mM

Swarming Assay

Capillary Assay

This assay didn`t check chemotaxis by E.coli. We did eleven assays. But all results weren`t authenticity.

Fig.1-A was result that plating Asp contained capillary. There were 50 colonies.

Fig.1-B was result that plating control contained capillary. There were 22 colonies.

Future work

Reference