Part:BBa_K4711041

NeuAc riboswitch+φ174E

Usage and biology

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.

During the validation process of this suicide switch, measuring OD values is often required. However, due to the excessive concentration gradients set, the procedure for sampling and measuring OD values becomes cumbersome. Therefore, we recommend using a 96-well plate or a 24-well plate for cultivation and measurement. However, when we actually measured using a 96-well plate, we found that the limited growth space and nutrient conditions in the small wells caused the Escherichia coli growth curve to shorten, leading to cell death within 3-4 hours. Therefore, we adjusted our sampling frequency from once every hour in the first experiment to once every half an hour in the second experiment. As a result, we recommend that future teams use a 24-well plate to ensure sufficient growth environment for Escherichia coli and achieve optimal data measurement.

Fig 1 (A) Gene circuit (B) Colony PCR (C) SDS-PAGE M: Protein Marker 1: 0mM IPTG 2: 0.2mM IPTG

Fig 2 OD600 changes after 0.2mM IPTG induction

Fig 3 OD600 changes after 0.5mM IPTG induction

References

[1] Wang, J. (2016). Evolution of N-acetylneuraminic acid producing recombinant Escherichia coli based on a riboswitch screening system. (Doctoral dissertation). Shandong University.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]