Difference between revisions of "Part:BBa K3408006"
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1. Experimental methods | 1. Experimental methods | ||
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1.1. Construction of the expression vector | 1.1. Construction of the expression vector | ||
The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of green fluorescent protein (GFP) and terminator of this device are synthesized by the biotechnology company according to the known sequence. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-P<sub>nar</sub>-GFP. | The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of green fluorescent protein (GFP) and terminator of this device are synthesized by the biotechnology company according to the known sequence. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-P<sub>nar</sub>-GFP. | ||
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Fig.1. The expression vector of device P<sub>nar</sub>-GFP | Fig.1. The expression vector of device P<sub>nar</sub>-GFP | ||
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1.2. Construction and screening of recombinant engineering bacteria | 1.2. Construction and screening of recombinant engineering bacteria | ||
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1.3. Characterization experiment | 1.3. Characterization experiment | ||
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Take 2 bottles of 50ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria. | Take 2 bottles of 50ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria. | ||
| − | ①Culture engineered bacteria which have been transformed successfully for 6 hours. | + | |
| + | ①Culture engineered bacteria which have been transformed successfully for 6 hours. | ||
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②Culture the test group and negative control in anaerobic and aerobic environment for 6 hours respectively. | ②Culture the test group and negative control in anaerobic and aerobic environment for 6 hours respectively. | ||
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③Use the fluorescence microscope to observe the presence of fluorescence in the test group and the negative control group. | ③Use the fluorescence microscope to observe the presence of fluorescence in the test group and the negative control group. | ||
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2. Expected results | 2. Expected results | ||
Revision as of 16:30, 18 October 2020
Pnar-B0034-GFP-B0015
We used promoter Pnar which was activated by FNR to trigger the expression of GFP(BBa_E0040). If there was no oxygen, then FNR could be transcribed and we could see green bacteria in our culture medium.
1. Experimental methods
1.1. Construction of the expression vector The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of green fluorescent protein (GFP) and terminator of this device are synthesized by the biotechnology company according to the known sequence. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector fragment to construct the recombinant expression vector pWB980-DB-Pnar-GFP.
Fig.1. The expression vector of device Pnar-GFP
1.2. Construction and screening of recombinant engineering bacteria
Using B. subtilis WB800N as the expression host, the secretion expression vector pWB980-DB was transformed by electro-transformation. Inoculate them on LB solid medium coated with 10 μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.
1.3. Characterization experiment
Take 2 bottles of 50ml LB liquid medium with 10 μg/mL kanamycin, and inoculate the same amount of recombinant engineered bacteria.
①Culture engineered bacteria which have been transformed successfully for 6 hours.
②Culture the test group and negative control in anaerobic and aerobic environment for 6 hours respectively.
③Use the fluorescence microscope to observe the presence of fluorescence in the test group and the negative control group.
2. Expected results
Fluorescence can be observed in the test group but not in the negative control group.
The negative control group The test group
Fig.2. Expected results: different expressions of fluorescence between the negative control group and the test group.
These results are predicted because of the lack of experiment for the COVID-19.