Part:BBa_K897720:Experience

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UNIQ755180bf3dd606d3-partinfo-00000000-QINU The antisense FtsZ, protected by hair-pin structure and termed TBY or BBa_897720, is regulated by T7 promoter and regarded as the core part of our project. The antisense FtsZ sequence mainly aims at silenting the FtsZ gene and control the self-renewal and differentiation of E.coli. Since the antisense FtsZ gene in BBa_897720 is regulated by the T7 promoter that is specifically combined with T7 RNA polymerase, a endogenous protein in BL21(DE3) strain under the control of the lac UV5 promoter inducible by IPTG, the number of colonies is expected to decrease with the an increase in IPTG. The confirmatory experiment below verifys the validity of TBY and our hypothesis.

After integrating the plasmid with an insert of BBa_897720 into BL21(DE3) strain, we cultivated those bacteria on the surface of solid LB medium in 4 dfferent IPTG levels. The conditions was strictly controlled at the temperature of 37 ℃ and it last for 10 hours. The controls aims to rule out the possibility of damaged to the bacteria from IPTG. Details are illustrated below:

	IPTG(0.0mM)	IPTG(0.5mM)	IPTG(1.0mM)	IPTG(2.0mM)
TBY *0.01
TBY *0.1
TBY- *0.1
TBY- *0.01

• TBY represents the BL21(DE3) bacteria carring plasmids with an insert of BBa_K897720. TBY- means that the BL21(DE3) bacteria carried the plasmids as same as the plasmids of TBY but without the insert of BBa_K8977720.
• TBY(-) are diluted tenfold and hundredfold separately, marked as “*0.1” and “*0.01”.
• The number in the parenthesis following the “IPTG” means the concentration of IPTG.
• For original full-size picture, [http://2012.igem.org/Team:SEU_O_China/Result/Pics see here].

In order to analyse the repressible effect of FtsZ, we calculate the colony coverage of every sample. Details are illustrated in the Model Part, and the results of analysis are shown below.

	IPTG(0.0mM)	IPTG(0.5mM)	IPTG(1.0mM)	IPTG(2.0mM)
TBY *0.01	500px
TBY *0.1
TBY- *0.1
TBY- *0.01

• Colony coverage Map

	IPTG(0.0mM)	IPTG(0.5mM)	IPTG(1.0mM)	IPTG(2.0mM)
TBY*0.01	93.23	9.21	0.65	0.29
TBY*0.1	76.30	44.38	27.74	19.37
TBY-*0.1	94.03	95.85	92.39	93.55
TBY-*0.01	80.01	82.51	86.74	88.97

• Percentage of colony coverage

• FtsZ repressible effect

Both of the red line and the blue line in the illustration show that the colony coverage decreases while the level of IPTG increases, which suggests that the growth of colony is somehow repressed. Moreover, the yellow line reveals that the IPTG has little side effect on colony. As a result, FtsZ truly represses the growth of bacteria and also provide experimental evidence for our project scheme.

UNIQ755180bf3dd606d3-partinfo-00000001-QINU