Team:BIT/Demonstrate
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Demonstrate
The following results show our project working under real-world conditions. We employed three ways to demonstrate our project working under simulated conditions in the lab including gel electrophoresis, sequencing and experiment test.
1. Live cell Logic gate genetic components
According to this picture, the NO.1 and No. 2 are J23104+B0034+Mir155 Binding site+Gfp (about 1200bp)
J23104(35bp)+B0034(12bp)+Mir155(97bp)+Gfp(878bp)+B0015(129bp)=1151(bp)
J23104(35bp) +B0034(12bp)+Mir21(90)+Rfp(706bp)+B0015(129bp)=972(bp)
So, the picture proves that that the length of our device is right. No.3 gene located on 30000bp is target gene (972bp) and backbone(2000bp).
Ptet(54bp)+B0034(12bp)+tetR(685bp)+B0034(129bp)+FimE(558bp)+B0015(129bp)+pLac(35bp)+B0034(129bp)+LacI(1125bp)+
B0034(12bp)+BXB1(1503bp)+B0015(129bp)+IRL+B0010+IRR+attB+B0010+attP(300bp)+B0034(12bp)+Gfp(878bp)+B0015(129bp)=5809(bp)
So, the picture prove that that the length of our device is right.(No.2,No.4,No.6)
Pconst(55bp)+B0034(12bp)+Mir21(90bp)+LacI(1125bp)+B0015(129)+Plac+key(plac and key 90bp)+Plac+Lock(Plac and Lock 126bp)+Gfp(878bp)+B0015(129bp)=1756(bp)
So, the picture prove that that the length of our device is right.(No.2,No.4,No.6)
Pconst(55bp)+B0034(12bp)+Mir21(90bp)+LacI(1250bp)+B0015(129)+Plac+key(plac and key 90)+Plac+Lock(Plac and Lock 126bp)+BXB1(1503) +B0015(129bp)=3384(bp)
So, the picture prove that that the length of our device is right.(No.2,NO.4)
2. Sequencing
We sequencing and alignment J23104-B0034-mi155-4-E0040-B0010-B0012
The identities are 99% right because of gene sequencing error and gene mutation. So this picture prove our device is right.
3. Experiment test
This is the growth curve of bacteria and from this figure, we can see that as time goes on, the bacteria reproduce which results in the rising concentration and OD value of bacterium. As for different experimental groups, all the groups with different concentration of mir155 have almost the same OD value, which indicate the growth of bacteria are synchronous during the detection of florescence. The factor of concentration of mir155 does not have strong impact on the growth of bacteria which accords with expectations of experimental results.
This figure show the fluorescence value generated by per unit bacteria volume which is also the experimental result of Part BBa_K2041021. When the mir155 presents in the system, mir155 will combine with the binding site. Meanwhile, the combination will influence the transcription and translation of downriver gene. Therefore, the expression of fluorescence will decrease and with the higher concentration of mir155, the expression of GFP will bring down gradually. After culturing for a while, fluorescence value generated by per unit bacteria volume will tend to a stable value.
This is the growth curve of bacteria and from this figure, we can see that as time goes on, the bacteria reproduce which results in the rising concentration and OD value of bacterium. As for different experimental groups, all the groups with different concentration of tetR have almost the same OD value, which indicate the growth of bacteria are synchronous during the detection of florescence. The factor of concentration of tetR does not have strong impact on the growth of bacteria which accords with expectations of experimental results.
For more details: https://2016.igem.org/Team:BIT/Biology
Contact Us
Beijing Institute of Technology,
No. 5 South Zhong Guan Cun Street,
Haidian Beijing 100081, P. R. China
Twitter : @igem_BIT
Sina Weibo : @igem_BIT
Website : http://www.bit.edu.cn
