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Light Sensing-Imaging System
Construction of Blue Light Sensing and Imaging System
Because of the complication of blue light circuit, work on this circuit began firstly. In the beginning, K592020 which is a blue light sensor output containing a FixK2 blue light sensing system and producing amilCP was constucted.
The culture of bacteria went like this:
And this is the bands exhibition of K592020 and K592016, another blue light sensor and RR with RBS, after electrophoresis, quite satisfied as we expected.
The electrophoresis result of other key parts in blue light sensing-imaging system including B0015, S04617 and K592024. The part I13502 which expresses mRFP was run with them.
Later, construction of the whole parts completed with validation of sequence verification.
Test of Blue Light Sensing and Imaging System
1.Test in solid media.
Without induced expression, bacteria containing blue light sensing-imaging system grew up and developed in the LB media. Then we divided the media into two regions, one of them receive blue light simulation while the other one keep darkness by a base plate to block blue light. The testing conditions showed in the following:
After 12 hours development, the result showed the bacteria could produce blue chromoprotein when stimulated by blue light and keep its color in the darkness.
2.Test in liquid media.
The idea of control-expriment is the same as in solid media. We prepared two test tubes containing equivalent bacterium solution. The test was conducted at 18:00, August 4th when one of them was put under blue light and the other was developed in the darkness.
At 11:00, August 5th, the tube stimulated under blue light turned light blue while the other one kept the original color.
After 24 hours, The blue bacteria showed evidently. Using centrifuge to get the bacteria showed extremely distinct blue chromoprotein expression base on blue light activation was come true.
Test of Green and Red Light Sensing-Imaging System
The procedure of experiment is the same as blue light test. The test started at 19:00, August 5th. After 22 hours growing up, bacteria after centrifuge appear red. but not obvious like blue and green. However, the GFP seemed not influence by light stimulation, which means it was also green in the darkness.
The whole result shows in the following, and 2 days later the OD600nm of all tubes is in the following as well.
Pattern Test
To test whether bacteria could produce a pattern with light stimulation, an experiment using projector to generate a pattern was conducted.
The pattern was this:
And the whole divice like this:
Because blue light sensing-imaging system works well in our project, we used it to produced a shadow in the media and then added other bacteria later, just like engraving the picture in the earliest photographing.
Calobacter crescentus
Conjugation
Conjugation protocol
conjugation of red light sensing-imaging system
verification of C.crescentus
We use Gram Staining to verify the C.crescentus:
- Prepare thin smear bacteria on a clean slide.
- Cover the smear with few drops of crystal violet for 1-2min.
- Wash excess strain with water.
- Add few drops of iodine solution on the smear for 1-2min.
- Rinse in water.
- Wash with 95% ethanol for 20s.
- Rinse in water.
- Stain with 1% safranin for 2-3min.
- Wash stain in excess water.
- Look at it under a microscope.
Growth Curve for Conjugation
To find the optimum of conjugation conditions, we test the development of S17-1 and C.crescentus in liquid PYE at 30 degree centigrade, which is almost the same condition while conjugation though the real conjugation is in solid PYE. We did so trying to get the result quantitively.
Motion Control
As we expected, DgrA, DgrB used for control the flagellum rotation and HfiA control the synthesis of lipid which is key component of holdfast.
The electrophoresis results like this.
DgrA ,DgrB and HfiA
