Part:BBa_K4609073
pET30a-araC-FRT-pBAD-HicA-FRT
Two plasmids, BBa_K4609073 and BBa_K4609074, were used in the system. HicA toxin and HicB antitoxin are expressed respectively. At low temperature, HicA toxin and HicB antitoxin are transcribed together and form a complex, which makes cells unable to grow normally and in a dormant state. When the temperature reaches 37℃, FLP gene regulated by CI857 thermosensitive promoter starts to be transcribed, and FLP will bind to FRT sites on both sides of HicA. The HicA gene expression system is knocked out so that E. coli can resume normal growth. At the same time, we can detect the normal expression of HicA system by inducing arabinose promoter. The system can play a role in regulating the growth of Escherichia coli.
The system mainly refer to the BBa_K3490030 (https://parts.igem.org/Part:BBa_K3490030), on the basis of the original system we will HicB expression system with FLP expression system integration in a plasmid, On the one hand, the plasmid we used was pET-30a, which was different from the PCP20 plasmid used in the original system and did not carry FLP gene itself. On the other hand, the difficulty of transfection with two plasmids was greatly reduced compared with that with three plasmids, which effectively improved the success rate of our plasmid transfection. In the previous design, HICA carrier was used for high copy and hicb for low copy, but we designed the same carrier, and achieved the same effect by adopting the method of high HICA concentration and low HICA concentration in the conversion process.
HicA toxin and HicB antitoxin are expressed respectively. At low temperature, HicA toxin and HicB antitoxin are transcribed together and form a complex, which makes cells unable to grow normally and in a dormant state. When the temperature reaches 37℃, FLP gene regulated by CI857 thermosensitive promoter starts to be transcribed, and FLP will bind to FRT sites on both sides of HicA. The HicA gene expression system is knocked out so that E. coli can resume normal growth. At the same time, we can detect the normal expression of HicA system by inducing arabinose promoter. The system can play a role in regulating the growth of Escherichia coli.
The system mainly refer to the BBa_K3490030 (https://parts.igem.org/Part:BBa_K3490030), on the basis of the original system we will HicB expression system with FLP expression system integration in a plasmid, On the one hand, the plasmid we used was pET-30a, which was different from the PCP20 plasmid used in the original system and did not carry FLP gene itself. On the other hand, the difficulty of transfection with two plasmids was greatly reduced compared with that with three plasmids, which effectively improved the success rate of our plasmid transfection. In the previous design, HICA carrier was used for high copy and hicb for low copy, but we designed the same carrier, and achieved the same effect by adopting the method of high HICA concentration and low HICA concentration in the conversion process.
1.Design
In order to make our products sleep during transportation and play an effective role when used by users. We constructed a two-plasmid system designed to regulate the growth state of E. coli DH5α. The system consists of two plasmids, E. coli resting plasmid PET30A-ARac-FRT-BAD-HicA-FRT, E. coli resuscitation plasmid pET30a-J23100-HicB-CI857-pR-FLP, and pET-30a vector for expressing HicA toxin and HicB antitoxin. At low temperatures, HicA and HicB are simultaneously transcribed and form complexes, causing cells to become dormant and unable to grow normally. However, when the temperature rose to 37°C, the CI857 thermosensitive promoter activated the expression of FLP recombinase, FLP bound to the FRT site around HicA, knocked out the HicA system, and allowed E. coli to resume normal growth. This system mainly referred to BBa_K3490030. Based on the original system, we fused the HicB expression system and FLP expression system into one plasmid, effectively improving the success rate of our plasmid transfection. In addition, arabinose-induced promoters were added to plasmid 4 to detect the normal expression of the HicA system. This system can effectively regulate the growth of Escherichia coli.
2.Build
In this part, we sought the help of qualified companies for plasmid construction, and the relevant companies provided us with bacterial solution with plasmids.
In order to ensure the correct sequence of constructed plasmids, we sequenced the plasmids, and the sequencing results were as follows:
Figure 1 Sequencing results of pET30a-araC-FRT-pBAD-HicA-FRT
Figure 2 Sequencing results of pET30a-J23100-HicB-CI857-pR-FLP
The results show that our design matches perfectly with the extracted plasmids, indicating that we have theoretically achieved the goal of the project.
3.Test
According to the design principle of the plasmid, due to the joint action of hicA and hicB plasmids, at 23¬°C (low temperature), our plasmid sleeps, and the growth of Escherichia coli is inhibited, but it can resume normal growth when placed at 37¬°C. Therefore, we set up two groups of bacterial solution, namely the control group (BL21 E. coli) and the experimental group (hicA-hicB plasmid co-transfer bacterial solution). After diluting the bacterial solution to an appropriate concentration, the plates of the two groups were coated. The plates of the control group were free of antibiotics, while the plates of the experimental group contained a certain concentration of kanamycin due to the resistance of the plasmid to kanamycin. Control group (cultured at 23¬°C), control group (cultured at 37¬°C), experimental group (cultured at 23¬°C) and experimental group (cultured at 37-°C) were put into the incubator. After 16 hours, the culture results were shown as follows: The bacteria in both the experimental group and the control group grew well under 37¬°C culture. The growth condition of the control group was also better at 23¬°C, while the experimental group at 23¬°C had few plate colonies, which proved that the dormancy system was basically effective. After observation, the plates cultured at 23¬°C in the experimental group were put into the incubator at 37¬°C, and the colonies increased significantly and grew well after 16 hours, which proved that the resuscitation system was also effective.
Figure 1 Co-transfer solution of hicA-hicB plasmid (cultured at 37 °C)
Figure 1 Co-transfer solution of hicA-hicB plasmid (cultured at 23 °C)
4.Study
After the experiment, we studied the original plasmid design team NCKU_Tainan again and found that they had done the kanamycin resistance gene deletion experiment, and the team constructed the pKD3 plasmid to prove the model, which carries the kanamycin resistance gene and is located between the two FRT sites. Bacteria rarely survived on LB plates with kanamycin after thermal activation to demonstrate that genes between FRT sites were deleted and antibiotic resistance genes were successfully deleted. We believe that we should also design corresponding experiments to prove the complete elimination of HicA system under 37 °C culture (or after plasmid thermal activation) in terms of principle, so as to more fully support our results. However, due to time reasons, our plan has not been implemented in time. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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