Difference between revisions of "Part:BBa K4711002:Experience"

 
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=Demonstration of the function of the lysis gene=
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'''Group''': [https://2023.igem.wiki/ouc-china iGEM Team OUC-China 2023]
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'''Author''': Qingyu Wu
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'''Summary''': Successfully demonstrated the lysis function of this gene
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In order to avoid the leakage of foreign genes into the environment, we construct a kill switch as shown in the figure below. Colony PCR successfully verified that the constructed plasmid was successfully introduced into the strain, as shown in the figure below, and a target band of about 750bp was obtained. SDS-PAGE after 5-6h induction by IPTG showed clear bands compared with the control group, but no clear bands were found in the induction group, because the induction group killed itself after expressing lytic protein, released hydrolase, and degraded all other proteins.
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After successful plasmid introduction and expression verification, NeuAc with different concentration gradients of 0g/L, 0.5g/L, 1g/L, 2g/L, 4g/L and 8g/L were added to the medium for culture, and OD values of E.coli BL21 with induction time were measured at different concentrations. To determine how much NeuAc should be added to the final medium to ensure the normal survival of E. coli. In the figure, 0h represents the time point of IPTG induction. It can be seen that E.coli BL21 in the control group and without IPTG induction presented S-shaped growth curves. The OD value of E.coli BL21 in the experimental group decreased with the decrease of NeuAc concentration at each concentration, because the less NeuAc concentration, the less NEUAC concentration, the less NEUAC concentration. This indicates that the less NeuAc binds to the riboswitch, the ribozyme cannot achieve self-shear, and the target gene behind it cannot be exposed and is hydrolyzed by the ReJ enzyme, so the lysed protein ΦX174 can be expressed in large numbers, causing the death of E. coli. This shows that our NeuAc ribose switch successfully performs its function. Through modeling analysis of the experimental data, we obtained that when NeuAc10g/L was added to the medium, Escherichia coli lysed and died after breaking away from the medium environment.
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        <img src="https://gitlab.igem.org/2023/ouc-china/-/raw/main/wiki/Photo/%E5%9B%BE%E7%89%871.png"width="80%" style="float:center">
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Fig 1. Gene circuit and colony PCR
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        <img src="https://gitlab.igem.org/2023/ouc-china/-/raw/main/wiki/Photo/%E5%9B%BE%E7%89%872.png" width="30%" style="float:center">
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Fig 2. SDS-PAGE M：Protein Marker 1：0mM IPTG 2：0.2mM IPTG
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<img src="https://gitlab.igem.org/2023/ouc-china/-/raw/main/wiki/Photo/%E5%9B%BE%E7%89%873.png" width="80%" style="float:center">
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Fig 3. OD600 changes after 0.2mM IPTG induction
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        <img src="https://gitlab.igem.org/2023/ouc-china/-/raw/main/wiki/Photo/%E5%9B%BE%E7%89%874.png"width="80%" style="float:center">
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Fig 4. OD600 changes after 0.5mM IPTG induction
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Latest revision as of 16:06, 21 September 2023


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Applications of BBa_K4711002

User Reviews

Demonstration of the function of the lysis gene

Group: iGEM Team OUC-China 2023

Author: Qingyu Wu

Summary: Successfully demonstrated the lysis function of this gene

In order to avoid the leakage of foreign genes into the environment, we construct a kill switch as shown in the figure below. Colony PCR successfully verified that the constructed plasmid was successfully introduced into the strain, as shown in the figure below, and a target band of about 750bp was obtained. SDS-PAGE after 5-6h induction by IPTG showed clear bands compared with the control group, but no clear bands were found in the induction group, because the induction group killed itself after expressing lytic protein, released hydrolase, and degraded all other proteins.


After successful plasmid introduction and expression verification, NeuAc with different concentration gradients of 0g/L, 0.5g/L, 1g/L, 2g/L, 4g/L and 8g/L were added to the medium for culture, and OD values of E.coli BL21 with induction time were measured at different concentrations. To determine how much NeuAc should be added to the final medium to ensure the normal survival of E. coli. In the figure, 0h represents the time point of IPTG induction. It can be seen that E.coli BL21 in the control group and without IPTG induction presented S-shaped growth curves. The OD value of E.coli BL21 in the experimental group decreased with the decrease of NeuAc concentration at each concentration, because the less NeuAc concentration, the less NEUAC concentration, the less NEUAC concentration. This indicates that the less NeuAc binds to the riboswitch, the ribozyme cannot achieve self-shear, and the target gene behind it cannot be exposed and is hydrolyzed by the ReJ enzyme, so the lysed protein ΦX174 can be expressed in large numbers, causing the death of E. coli. This shows that our NeuAc ribose switch successfully performs its function. Through modeling analysis of the experimental data, we obtained that when NeuAc10g/L was added to the medium, Escherichia coli lysed and died after breaking away from the medium environment.



Fig 1. Gene circuit and colony PCR

Fig 2. SDS-PAGE M：Protein Marker 1：0mM IPTG 2：0.2mM IPTG

Fig 3. OD600 changes after 0.2mM IPTG induction

Fig 4. OD600 changes after 0.5mM IPTG induction