Difference between revisions of "Team:Nanjing-China/Notebook"
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<div class="word" id="protocol"> | <div class="word" id="protocol"> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| − | <h3> | + | <h3>ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd<sup>2+</sup> adsorption</h3> |
| − | + | <p><em>Escherichia coli</em> BL21 containing <Em>OmpA-PbrR-PJQ200SK</Em> (pBAD33) plasmid was cultured in LB medium to an OD<sub>600</sub> of 0.4-0.6. Arabinose and CdCl<sub>2</sub> were added to the medium to a final arabinose concentration of 40 μM and a final Cd<sup>2+</sup> concentration of 100 μM, to induce the formation of CdS nano semiconductors.<br /> | |
| − | + | From the start of the induction, 5 ml of the bacterial solution was taken from the culture every 6 hours (sampling to 24 hours), centrifuged at 4000 rpm for 2 minutes, and washed three times with water to remove the medium involved in the bacterial surface. <br /> | |
| − | + | The washed bacteria were resuspended in 5 ml of water. OD<sub>600</sub> was measured, and the bacteria were collected by centrifugation.<br /> | |
| − | + | 3 ml of concentrated nitric acid was added and the mixture was digested overnight at 90 °C.<br /> | |
| − | + | The Cd<sup>2+</sup> content in the sample was measured using ICP-MS.</p> | |
| − | + | <h3>Cd<sup>2+</sup> toxicity test</h3> | |
| − | + | <p>Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl<sub>2</sub> were added to the medium to form a Cd<sup>2+</sup> gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.<br /> | |
| − | + | <em>E. coli</em> BL21 containing the <em>OmpA-PbrR-PJQ200SK</em> (pBAD33) plasmid and plasmid-free <em>E. coli</em> BL21 (control) were cultured in different media.<br /> | |
| − | + | The OD<sub>600</sub> value was measured every 2 hours and measured for 12 hours.</p> | |
| − | + | <h3>Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX)</h3> | |
| − | + | <p>After the Cd<sup>2+</sup> adsorption induction was completed, the bacteria were collected by centrifugation and resuspended in ultrapure water. Samples were sent for TEM image acquisition.<br /> | |
| − | + | The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV. </p> | |
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<div class="word" id="reference"> | <div class="word" id="reference"> | ||
<h2>Reference</h2> | <h2>Reference</h2> | ||
Revision as of 03:09, 13 October 2018
MAY
Protocol
ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd2+ adsorption
Escherichia coli BL21 containing OmpA-PbrR-PJQ200SK (pBAD33) plasmid was cultured in LB medium to an OD600 of 0.4-0.6. Arabinose and CdCl2 were added to the medium to a final arabinose concentration of 40 μM and a final Cd2+ concentration of 100 μM, to induce the formation of CdS nano semiconductors.
From the start of the induction, 5 ml of the bacterial solution was taken from the culture every 6 hours (sampling to 24 hours), centrifuged at 4000 rpm for 2 minutes, and washed three times with water to remove the medium involved in the bacterial surface.
The washed bacteria were resuspended in 5 ml of water. OD600 was measured, and the bacteria were collected by centrifugation.
3 ml of concentrated nitric acid was added and the mixture was digested overnight at 90 °C.
The Cd2+ content in the sample was measured using ICP-MS.
Cd2+ toxicity test
Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl2 were added to the medium to form a Cd2+ gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.
E. coli BL21 containing the OmpA-PbrR-PJQ200SK (pBAD33) plasmid and plasmid-free E. coli BL21 (control) were cultured in different media.
The OD600 value was measured every 2 hours and measured for 12 hours.
Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX)
After the Cd2+ adsorption induction was completed, the bacteria were collected by centrifugation and resuspended in ultrapure water. Samples were sent for TEM image acquisition.
The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV.
Reference
- Kathryn RF,Yanning Z,et.al.(2016)Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium PNAS
- Wang L,Zhang L,Liu Z,Zhao D,Liu X et.al(2013) A minimal Nitrogen Expression of Active Nitrogenase in Escherichia coli PLOS Genetics9(10):e1003865
- Katherine AB,Derek FH,Molly BW et.al(2016) Light-driven nitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid Science352,448
- Wei W,Sun PQ,Li Z,Song KS,Su WY,Wang B,Liu YZ,Zhao J et.al (2018) A surface display biohybrid approach to light-driven hydrogen production in air Science eaap9253
- Wei W,Zhu T,Wang Y et.al(2012) Engineering a gold-specific regulon for cell-based visual detection and recovery of gold Chem.Sci,3,1780-1784
- James BH&Douglas CR(1996) Structural Basis of Biological Nitrogen Fixation Chem.Rev.96,2965-2982
Address
Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046
