Team:XMU-China/Improve
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This year, XMU-China still devotes to making improvements for previous bricks according to our project. Our improved parts are INPNC(BBa_K3332016) and pBLind promoter.
Background
Ice nucleoprotein is an anchor protein from Pseudomonas syringae. It can anchor its passenger protein to the cell membrane. N and C terminal of ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane. SpyTag is a peptide fragment, which could steadily bind to SpyCatcher by forming an isopeptide bond.
Design
This season, we have improved the function of INPNC expressed by the gene in brick of BBa_K3332016. The INPNC-GFP protein expressed by BBa_K3332016 could make us confused whether the fluorescently derived from cytoplasm or cytomembrane of E. coli. It is also tricky to verify whether a protein has been successfully displayed on the surface of E. coli. Thus, we constructed two fusion proteins of INPNC-HisTag-SpyCatcher (BBa_K3739102) and HisTag-SpyTag-GFP (BBa_K3739103), which make the display of INPNC more visualized and efficient (Fig. 1). The fused protein of INPNC-HisTag-SpyCatcher, successfully displayed on the surface of E. coli by INPNC, could bond with the HisTag-SpyTag-GFP protein externally added and form an isopeptide. The green fluorescence in HisTag-SpyTag-GFP could be directly observed by using the fluorescence confocal microscope.
Fig. 1. Schematic diagram of the bonding process between INPNC-HisTag-SpyCatcher and HisTag-SpyTag-GFP.
Result
1mL E. coli BL21(DE3) culture solution, which has express fusion membrane protein of INPNC-HisTag-Spycatcher, was used for the following experiment. Another engineered E. coli BL21(DE3) with HisTag-Spytag-GFP was also cultured, and being harvested in sediment after being centrifuged with the condition of 8000 g, 10 min, and 4°C. After broken and centrifuged, the supernatant solution with HisTag-Spytag-GFP was obtained and incubated with the E. coli BL21(DE3) culture solution (express INPNC-HisTag-Spycatcher) at 37 °C in the shaker. Samples were taken every two hours. After centrifugation, the fluorescence of 50 μL supernatant of each sample was measured (λex = 475 nm, λem = 545 nm), in which the bacteria incubated with PBS solution was set as control. At the same time, the fluorescence confocal microscope has also been employed to verify the bonding of HisTag-SpyTag-GFP to INPNC-HisTag-SpyCatcher (Fig. 2). After incubation, the decrease of fluorescence intensity of supernatant indicated the binding of HisTag-SpyTag-GFP to the INPNC-HisTag-SpyCatcher displayed in the surface of E. coli BL21(DE3), while the fluorescence intensity in the control group changes slightly (Fig. 3).
Fig. 2. The bonding of INPNC-HisTag-SpyCatcher to HisTag-SpyTag-GFP was observed from the fluorescence confocal microscope. Scale bar, 5 μm.
Fig. 3. Time course of fluorescence changes of the supernatant.
Background
As a light-regulated module, the pBLind promoter can be activated by the blue light dependent DNA-binding protein EL222. However, given that the blue-light induced system suffers from a considerable leakage of expression in dark environment, especially when EL222 is constitutively expressed, we attempted to find a way to improve the promoter system in this year’s project.
Design
In some designs of pBLind-EL222 system, EL222 was usually expressed by a constitutive promoter in the Anderson family, such as J23119 and J23106. Although the EL222 protein is activated to bind pBLind promoter due to the conformational change upon blue light illumination, the binding events still happen by chance in dark environment, which results in unexpected transcriptional leakage of the genes controlled by pBLind promoter. Reducing the existent pool of EL222 protein in the cell will low down the leakage level in the dark state. Therefore, using an inducible promoter to control the expression of EL222 seems to be an ideal option to lower the leakage when the blue-light irradiation is not supplied.
Fig. 4. Gene circuit illustration for improved pBLind promoter system.
For some inducible systems, such as arabinose-inducible system or rhamnose-inducible system, small molecules (i.e. inducer) are added into the culture to induce the corresponding promoter of the system. Although chemical induction is commonly used, we try to find alternative means to induce the expression of EL222 without introducing any chemicals to our system. With this intention, we used pBLind promoter (BBa_K2332002) to replace the constitutive promoter controlling the expression of EL222 and chose sfgfp (BBa_K2560043) as the reporter gene, thus created pBLind-EL222-pBLind-sfgfp (BBa_K3739064) (Fig. 4). We speculated that due to the lower level of leakage in dark environment, a higher dynamic range for downstream gene’s (sfgfp) expression controlled by this light-induced system might be obtained.
Result
Fig. 5. The leakage expression and light sensitivity of the modified light-induced system (BBa_K3739064) was characterized. (A) RFUsfGFP/OD600 of modified and original system was calculated as time progressed. (B) The dynamic range of the two systems was compared by the RFUsfGFP/OD600 at the last time-point of measurement.
First, we characterized the leakage of the original promoter system (left in Fig. 4) and the modified one (right in Fig. 4) by measuring the fluorescence intensity (λex = 488 nm, λem = 530 nm) and OD600 under strictly shadowed condition. The relative fluorescent unit (RFU) normalized to OD600, RFUsfGFP/OD600, was calculated to represent the expression level of sfGFP in an average cell.The modified system showed a lower expression level of sfGFP in the dark condition, while the original system indeed had a higher leakage level during the whole measuring time (Fig. 5A). To verify that the light-induced system can still function well when exposed to blue-light illumination, we then implemented another measurement to characterize the photoensitivity of J23106-EL222-pBLind-sfgfp and pBLind-EL222-pBLind-sfgfp under consistent blue-light irradiation. Surprisingly, the modified system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the modified system was observed as time progressed (Fig. 5A). Due to the lower leakage level in the dark condition and higher expression level upon blue-light irradiation, the modified system had a higher dynamic range (~6.9-fold) than the original system (~4.5-fold), in which the EL222 protein is constitutively expressed (Fig. 5B).
In conclusion, we constructed a modified light-induced system with lower leakage expression and improved light sensitivity. It should be noticed that the experiments mentioned above were all performed in E. coli, the most widely used chassis in synthetic biology. Actually, we had tested the modified system in Vibrio natriegens and similar phenomena was observed (see our Result page). The results directly benefited the implementation of our kill switch system (see our Proof of Concept page), and we hope this improved design would inspire those who plan to use pBLind promoter (BBa_K2332002) and its regulator EL222 (BBa_K2332004) in their gene circuits.