Team:ZJUT-China/Experiments
ZJUT-China
Experiments
Our experiments include all four parts: the test of light-controlled system, the test of functional gene lysin, the combination of two parts, and the test of the whole system.
At first we used the plasmid from Shanghai Jiao Tong University which used mRFP as reporter gene. For convenient test, we replaced mRFP with eGFP ,which can be lnminated in UV. But when we tested, we found the fluorescence intensity of experimental group with light sensing system was obscure compared with the control group without it. We then added a strong constitutive promoter BBa_J23100 before YF1 but with little help.
Light-controlled System Test
Referring to previous thesis, we found the FixK2 promoter reported in the thesis was different from we used for light sensing system:BBa_K592006. We finally decided to synthesize a plasmid with primitive FixK2 promoter, we also used BBa_J23100 promoter and added a terminator before FixK2 promoter. The circuit is as it shows in the picture.
- The result is shown in the figure below:
- Figure 1: The result of light-controlled system test
Lysin test
Lysin was directly synthesized and was tested in pGLO, regulated by araBAD promoter. We tried to gain the lysis rate by testing OD600. But then we found it difficult to get an accurate lysis rate though this way. So we changed to dilution-spread method and gained the lysis rate successfully.
The results of OD600 testing and dilution-spread method are shown in the figures below.
②
Figure 2: ①The results of OD600 testing ;②Spread plate method results
Combination
After testing two parts of the whole system, we started to combine to parts. All two parts and linearized vector were prepared by PCR and connected by One Step Cloning after cleaning up.
Final Test
The completed recombined plasmids were transferred into E.coli BL21 (DE3), and tested under blue light induction.
The result is shown in the figure below.
Figure 3: Final test of recombined systerm results
Important Protocols
LB medium (solid, 1L = 50 dishes) :
- 2 g / 100ml (in the conical flasks)
- 10 g tryptone
- 5 g yeast extract
- 5 g NaCl
- Water
