The plasmid containing the CsgA-Mfp3S-pep fusion protein gene was first synthesized,transferred into the BL21 (DE3) receptor state, and subjected to colony PCR. the resulting.DNA electrophoresis bands are shown below.
Fig.1 CsgA-Mfp3S-pep fusion protein bacteriumColony PCR DNA electrophoresis band diagram
The successfully transferred bacteria were divided into two groups, one with IPTG and one without IPTG, and the induction of expression was started in M63 broth medium at an OD600 of about 1. Four days later, a dish with IPTG and a blank control dish of amilcp bacteria were stained with crystalline violet, and the staining results are shown below
Fig.2amilCP bacteria Colony PCR DNA electrophoresis band diagram
Its band of about 800bp represents a successful transfer. PCR was performed on the CsgA-Mfp-pep fragment, and its PCR product was purified by DNA product and then double digested, and its DNA electrophoresis bands were as follows
Fig.3 CsgA-Mfp3s-pep fragment PCR DNA band map
The band is about 750bp, which is the target fragment we want. The amilCP plasmid was double digested and the DNA electrophoresis bands were as follows
Fig.4 amilCP plasmid double digestion DNA band map
The products were subjected to DNA purification product recovery and the recovered fragments were ligated with T4 ligase, respectively. The resulting plasmids were transferred into DH5α receptor state and then subjected to colony PCR with the following DNA electrophoresis bands.
Fig.5Synthetic Plasmid Bacteria Colony PCR DNA strip map
The band size of about 1500bp represents successful transfer.
The bacteria transferred into amilCP plasmid were divided into two groups, one with IPTG and one without IPTG while expression was induced at an OD600 of about 1. The induction results were as follows
Fig.6amilCP graph of induction expression results
The bacterium in the right tube in the figure has a clear blue phenotype compared to the bacterium in the left tube, which proves that the induction system can function as expected.
The bacteria transferred into CsgA-Mfp3S-pep plasmid were divided into two groups, one with IPTG and one without IPTG while expression was induced in M63 broth medium starting at an OD600 of about 1. Three days later, a dish with IPTG and a blank control dish of amilCP bacteria were stained with crystal violet, and the staining results are as follows
Fig.7 Crystalline violet staining results
Their staining results demonstrated the presence of biofilm formation and the effect of CsgA with Mfp3S. Mineralization of the other unstained biofilms was performed. The SBF was changed every two days for 7 days of mineralization. samples from day 0 and day 7 were sent for scanning electron microscopy and the results were as follows.
Fig.8 SEM image showing the surface morphology of the biofilm (i.e., unmineralized). Scale bars: 10μm (left)3μm (right)
Fig.9SEM image showing the surface morphology of the mineralized composite (mineralization 7d). Scale bars: 10μm (left)3μm (right)
Figure 7 shows day 0 and Figure 8 shows day 7. It can be observed that the samples on day 0 are still in the biofilm indicated state (i.e. unmineralized) the samples on day 7 show the surface morphology of mineralized composites (i.e. mineralized) showing the success of CsgA-Mfp3S-pep fusion protein expression and mineralization.
The plasmid containing the photosensitive system gene was first synthesized and transferred into the DH-5α receptor state together with the CsgA-Mfp3S-pep-amilCP plasmid. The bacteria were inoculated 1:100 in two groups of fresh LB, one group completely wrapped in tinfoil to avoid light and the other group was illuminated using a blue LED light. The results after two days of simultaneous incubation and centrifugation at 12,000 rpm for 1 min were as follows
Fig.10(Left) Light culture (Right) Sheltered culture
It can be clearly observed that the bacteria on the left side of the light culture appear a distinct blue phenotype compared to the bacteria on the right side of the light-proof culture. The experimental results show that the light-sensitive system can function as expected.
The photoreceptor system-T7RNAP plasmid was transferred into DH-5α receptor state along with CsgA-Mfp3S-pep-amilCP recombinant plasmid. The bacterium was inoculated 1:100 in two plates of M63 broth medium, and one plate was irradiated using a light-proof cloth wrapped around the Petri dish and the other plate was irradiated using a blue LED light, and mineralized after three days of induction culture. The mineralization results were as follows
Fig.11 (Left) Mineralization results of blue light irradiation culture (Right) Mineralization results of light-proof culture
The results demonstrate the success of amilcp-CsgA-Mfp3S-pep in combination with the blue light sensing system, the success of our experiments and the feasibility of our project.
To reduce the amount of leaked expressions. We were inspired to apply it in our project. Referring to the structure of BL21(DE3)+pET28 protein expression system, we wanted to make both T7 promoter and T7RNAP under the control of PhlF to reduce the leakage activity, so we added PhlO sequence after the T7 promoter. We attached BBa_K3254024 elements downstream of both the original T7 promoter and the modified T7 promoter with PhlO binding site to characterize the strength of the promoter.
Fig.12Diagram of PhlO device
We attached each of these two test devices to the pSB4C5 backbone and transformed them into an E. coli that constitutively expresses T7RNAP.1 The experimental results showed that this modification did not significantly affect the activity of the T7 promoter (compare the blue column in the figure below). Subsequently, we transferred to this system the BBa_K2525007 element attached to the pSB1K3 backbone. We inserted the strain into liquid LB medium and incubated it overnight. Bacterial broth 1:200 was inserted into fresh LB medium (containing IPTG) and incubated at 37 degrees C, 220 rpm for 8 hours. 200 µl of culture was taken into 96-well plates and 485/510 fluorescence and OD600 were measured using an enzyme marker. sfGFP expression was expressed as the ratio of fluorescence value to OD600.
Fig.13PhlO and PhlF assay results
We plotted the results in the above figure. The experimental results showed that PhlF significantly inhibited the activity of pT7-phlO by more than 70-fold.
1. Zong, Y., Zhang, H. M., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q., and Lou, C. (2017) Insulated transcriptional elements enable precise design of genetic circuits, Nature communications 8, 52.
