Difference between revisions of "Team:OUC-China/Project/Captor"

How come?

For easy measurement of magnetism

If one told you that he’s going to measure the magnetism of some materials, he means that he’s going to measure magnetic behaviors of those materials, because “magnetism” holds a wealth of meaning. For examples, the susceptibility, magnetization curves for fields and hysteresis loop. The quantitative characterization of all magnetic behaviors mentioned above require a special instrument: The superconducting quantum interference device(SQUID). Fortunately, Pro. Pan and Pro. Cao from INSTITUTE OF GEOLOGY AND GEOPHYSICS，CAS kindly offered us help for quantitative characterization of the magnetic properties of our E.coli after induction and mineralization.

However, SQUID is not accessible for all labs. Researchers interested in bio-mineralization have to pay much for renting SQUID(¥260/h). And in many occasions, there’s no need for researchers to get the specific magnetic properties(for example, choose the best mineralization culture), just comparing the “strength of magnetism”(This expression is what we always think of, if measure the specific property, “specific magnetic moment” is a more scientific expression) is enough.

During processes to test magnetism of E.coli, we found that: If we set a magnet under the plate for a while, mineralized E.coli will gather at the edge of the magnet. And the shape of the magnet formed by well-mineralized E.coli is much more obvious than weak-mineralized E.coli. This interesting discovery inspired us to develop a method for easy measurement of magnetism: compare the shape conformed by different mineralized E.coli to compare the “strength of their magnetism”.

Fig.1. shape formed by weak-mineralized E.coli

Fig.2. shape formed by well-mineralized E.coli

When comparing many replicates, we found it inconvenient to take photos of every plate under the same condition. Thus, we designed a device to place a 6-well plate and 6 same magnets to make this measurement process more convenient.

Since this device is designed to catch magnetic E.coli, we named it “Captor”.

For convenient test of optimal inducement

When testing best induce concentration for PBAD on agar, we incubated culture at 6 different concentrations of arabinose, each concentration has 3 replicates. Then we met the same trouble: It’s inconvenient to take photos of so many replicates under the same condition, especially the condition with excitation light.

Naturally, we thought of “Captor”, designed for convenient and easy measurement of magnetism. And we successfully integrated the function of testing optimal induce concentration into Captor.

Fig.3. How we compare magnetism strength

Fig.4. How we took photos of plates with fluorescence before

Fig.5. How we compare induce concentration before

How to construct ?

Our Captor consists of 5 parts: box, lid, shelf, bottom and drawer. Here are their functions:
Box: light blocking
Lid: dust prevention
Shelf: 6-well plate supporting
Drawer: magnets supporting
Bottom: electric circuit supporting

Fig.6. Components of Captor

Fig.7. Appearance of Captor

Fig.8. 3D design of Captor

We designed a blueprints, and contacted company to produce it using acrylic. Click here to download blueprints

For magnetism measuring part:

We fixed 6 same magnets on a 6-well plate. This 6-well plate was supported by 2 rubber bands, and was set in the drawer. Shelf can fit 6-well plate in the proper location. While testing, another 6-well plate will be set on the 6 magnets.

Fig.9. The 6 fixed magnets

Fig.10. The 6 fixed magnets in box

For fluorescence measuring part:

We fixed 12 radiators on the bottom, and then set the LED beads(480~490nm and 450~455nm) on radiators. Weld the wire, LED beads and a three-way switch. The electric circuit is supported by a 12V Power Adapter.

Fig.11. Set LED beads on bottom

Fig.12. The electric circuit

How to use ?

For magnetism measuring part:

Step1: Take out 20mL bacterium solution per flask and centrifuge on 8,000g;
Step2: Discard the supernatant with PBS for 3 times to wash off the metal ions avoid false positive;
Step3: Resuspend the bacterium within 2mL PBS;
Step4: Add 300uL of the above-mentioned liquid into one plate of the 6 well plate of the Captor whose bottom has been blacked.
Step5: 1mL LB liquid medium and 1mL PBS are added in the plate. Shake and blend it softly. Take a picture of the 6 well plate now as the initial base for Matlab analyzing;
Step6: The rest of the liquid(about 2.1mL) is added into the disposable plate with 10mL PBS and 2mL LB liquid medium. Shake and blend it softly.
Step7: Put a piece of black paper between the plate and the magnet.(It can be omitted if the background is black)
Step8: After 12 hours, take a picture of the 6 well plate again as the post-experiment result coupled with the initial base for Matlab analyzing.

Step1: Use a 300*300px marquee to select the region contains the shape;
Step2: Use imread function to get the gray level;
Step3: Calculate the average background, and minus background from gray level;
Step4: Use mesh to output the plot.

Fig.13. Analyze of well-mineralized E.coli

Fig.14. Analyze of weak-mineralized E.coli

For fluorescence measuring part:

Step1: Prepare a 6-well plate containing 2mL LB agar per well with appropriate concentration of antibiotics;
Step2: Add 120μL culture of target E. coli, whose OD value is about 0.3, into each well and incubate it (37°C) for 2h;
Step3: Put sterile oxford cup in the center of each well, then add 40μL different concentration of arabinose into each cup;
Step4: Move it into constant temperature incubator (37°C) carefully.
Step5: Incubate it overnight.
Step6: Remove the oxford cup and observe the GFP in the Captor.

We found that it is hard to take fluorescent photo by Digital Single Lens Reflex because of the blue light.

To solve this problem, we use photoshop to process the image. We adjust the RGB parameter to make it in low blue value and high green value. Then we enhanced contrast ratio and luminance. Finally, we obtained the following image, which can be easily seen the green circles. The extent of the green is consist with the promoter strength.