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Lab Notes

The Lab Notes part only shows some of the intermedia raw data of our project. For final demonstrate, please move to the Demonstrate page provided below:

https://2019.igem.org/Team:HZAU-China/Demonstrate

Verification of the erasure module

Figure 1. Microplate reader raw data from the experiment of the verification of the erasure module.

Figure 1 is the microplate reader data of the demonstration of the erasure module. After induced for 8 hours, 100ng/mL ATc was added to the experimental group (+ATc) to transcribe AiiO. Fluorescence intensity (Excitation: 581nm, Emission: 607nm) and OD₆₀₀ were measured by a microplate reader. The first column was the group without ATc (Anhydrotetracycline) added, the second column was the group with ATc added. The curves describe the intensity of fluorescence over time. Row A, B and C are the three different biological repeats. The results show that the fluorescence intensity of the group we induced (Line 2) is much lower than the group uninduced (Line 1). We can infer that the erasure module has worked.

Verification of the switch in the memory module

Figure 2. Microplate reader raw data from the experiment of the verification of the switch in the memory module.

Figure 2 is microplate raw data of the demonstration of the switch in the memory module. After inducing for 8 hours, 100ng/mL ATc was added to the experimental group (+IPTG). Fluorescence intensity (Excitation: 485nm, Emission: 528nm) and OD₆₀₀ were measured by a microplate reader. The first column was the group with IPTG supplementation, the second column was the group without IPTG supplementation. The curves describe the intensity of fluorescence over time. Row A, B and C are the three different biological repeats. The curves of the first column tend to arise and there is no obvious upward momentum of the second column. This phenomenon indicates that the switch of the memory module can work.

Verification of the smell sensing module

Figure 3. PCR results of the construction of the smell sensing module.

Figure 3 is the PCR result of the construction of the smell sensing module. From left to right: A. Line 1 is marker, Line 2 and 3 are pSB1C3 vector backbone repeats and Line 4 and 5 are areR-P_are fragments. B. Line 1 is marker, Line 2 and 3 are gfp-his fragments. C. Line 1 is marker, Line 3, 5 and 6 are areR-P_are-gfp fragments. The results show that the benzyl alcohol sense module was successfully constructed.

Figure 4. Verification of the smell-sensing module under fluorescence microscope. E. coli with smell-sensing module were inspected under the fluorescence microscope after introduced by benzyl alcohol for 8h. A1. and A2. are experimental groups with benzyl alcohol added. B1. and B2. are control groups without benzyl alcohol added. The experiment results show that the smell-sensing module could produce GFP after being induced by benzyl alcohol.

Figure 4 demonstrates the results of the verification experiment of the smell-sensing module under fluorescence microscope. The bacterial cells with benzyl alcohol induced for 8 hours was observed with white light and blue light respectively (Figure 4A1, Figure 4A2), and obvious green fluorescence can be observed under blue light. The bacterial cells without benzyl alcohol induced was observed with white light and blue light respectively (Figure 4B1, Figure 4B2), and no green fluorescence could be detected under blue light. The experiment results show that the smell-sensing module could produce GFP after being induced by benzyl alcohol.

Construction of the memory module

Figure 5. Four fragments required for constructing the plasmid of memory circuit From left to right: linepSB1C3 is the linearized pSB1C3 vector backbone; luxpR::GFP:LVA is the lva-tagged gfp gene driven by luxPR ; luxpR::luxI is the luxI gene driven by luxPR promoter; J23102::luxR is the luxR gene driven by J23102.

Figure 6. Intermedia plasmid of the memory module. Left: pSB1C3-Memory is the intermedia plasmid used in memory module. Right: 5000bp-length marker. The right construction was confirmed by DNA sequencing

Construction of the plasmids used in parts improvement

Figure 7. PCR results of the improvement of part BBa_R0062. From left to right: Line 1 to 8 are seven different mutants of BBa_R0062, Line 9 is luxR fragment. Line 10 is sfgfp fragment. Line 11 is a backbone with ampicillin resistance and p15A ori. Line 13 is a 2000bp-length marker. The fragments were cloned to the backbone by homologous recombination. The right constructions were finally confirmed by DNA sequencing.

Construction of the plasmid used in part characterization

Figure 8. Construction of the plasmids used in part BBa_C0062 characterization. A. Line 1 and Line 3 are segments containing promoter, RBS and terminator. Line 2 is a 2000bp-length marker. B. Line 1 and Line 3 are linear pSB1C3 backbones containing luxPR, RBS, gfp and terminator. Line 2 is a 5000bp-length marker. C. From left to right, the first line is a 2000bp-length marker. The last line is the negative control. Other lines are positive results. The right constructions were confirmed by DNA sequencing.

Verification of tet operon

Figure 9. Fluorescence microscope detecting sfGFP in E. coli. A&B. The bacterial cells with ATc added was observed with white light and blue light respectively. C&D. The bacterial cells without ATc added was observed with white light and blue light respectively. (All the final concentration of ATc is 50ng/ml.)

Figure 9 demonstrates the results of tet operon which is used in the erasure module. The bacterial cells with 50ng/mL ATc induced for 2 hours was observed with white light and blue light respectively (Figure 9A&B), and obvious green fluorescence can be observed under blue light. The bacterial cells without ATc induced was observed with white light and blue light respectively (Figure 9C&D), and no green fluorescence could be detected under blue light. The result shows that tet operon can work well and will serve the erasure module.

Construction of the reproduction module

Figure 10. Colony PCR result for the benzyl alcohol synthesis circuit. Line 1 is 2000bp-length marker, the others are the results of different colonies. The result shows that the colonies in Line 2, 4, 6, 7 and 9 may have been successfully constructed.The right constructions are finally confirmed by DNA sequencing.