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E. coli Photography
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, 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, were quite satisfying 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 received blue light simulation while the other one kept darkness by a base plate to block blue light. The testing conditions are showed in the following:
After 12-hours' development, the result showed the bacteria could produce blue chromoprotein when stimulated by blue light and kept its color in the darkness.
2. Test in liquid media.
The idea of control experiment is the same as that ain 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 based on blue light activation was come true.
Test of Green and Red Light Sensing-Imaging System
The procedure of experiment is the same as that of 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 influenced 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: a computer producing pattern, a projector which converts electric signal into light signal and an imaging box. Medium is put into the dark box and the whole process includes about 1 day.
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.
Test of a sun pattern focused on sharp shape of a picture is in the following. The result showed bacteria could produce protein at the edge of the picture accurately.
C. Imager
Conjugation
Conjugation of red and blue photography system
After thousands of rounds of tests and condition improvements, red and blue light photography system has been successfully tranferred into C.crescentus using conjugation. The results are as following:
Blue conjugation
Protein electrophoresis also proved expression of amilCP.
Red conjugation
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 curves for conjugation
To find the optimum of conjugation conditions, we tested the development of S17-1 and C.crescentus in liquid PYE at 30 degree centigrade separately, 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, from which we extracted values of parameters in conjugation modeling.
Adhesion test of C.crescentus
To prove the indication in papers that C.crescentus's holdfast is able to adhere to surfaces strongly, we tested the adhesion ability of C.crescentus compared with E.coli
We firstly cultivated C.crescentus and E.coli in their best growth condition. Several slide glasses were put into the media to let bacteria adhere to glass surface in enough time. After one-day's development, slide glasses were picked out and washed in a strong and stable water flow about 2 mins. Then using Gram Staining, we could detect the bacteria on the glasses surfaces. The results showed that much more C.crescentus existed on the surface than E.coli, which proved enough adhesive ability of C.crescentus. More meticulous experiments should be conducted under electromicroscopes.
Motion Control
DgrA, DgrB are used to control flagellum rotation and HfiA inhibits the synthesis of lipid which is a key component of holdfast.
The electrophoresis results is like this.
DgrA, DgrB and HfiA
The standard parts construction have been accomplished, which helps motion regulation and holdfast biosynthesis of bacteria.
Kill Switch
We constructed the plasmid with lactose operator and signal peptide, and transformed this plasmid into E.coli to test if this kill switch works. We used gradient plate method in our experiment. As we can see in the picture, the concentration of IPTG on the left of the medium is 0.1% while the concentration on the right is zero. We can see colony only on the right of the medium, the kill switch is effective which illustrates that IPTG can induce the expression of signal peptide and LALF so as to inhibit the growth of the E.coil. Also, a decrease of OD is observed too, after we added IPTG to the liquid culture medium, which means our kill switch did work well.
