Part:BBa_K5048014
T7-lac-Lpp'OmpA(codon optimized)-DDDDK-AQ-GST-dt
This part is the surface-decorating system which displays small peptides AQ in the outer leaflet of the outer membrane of E.coli. The DDDDK enterokinase cleavage site can be recognized by enterokinase and small peptides will be cut off. It contains the T7 promoter, Lac operator, Lpp'OmpA(codon optimized), enterokinase site, AQ, GST tag, rrnB T1 terminator, and T7Te terminator. It is used in the verification of the enzymatic cleavage system.
Background of the Lpp’OmpA
Lpp’OmpA is a chimera developed by Georgiou and co-workers consisting of the signal peptide and the first nine residues of Braun’s lipoprotein or Lpp (Lpp’), responsible for the targeting to the outer membrane, fused with five of the eight membrane-spanning segments of the OmpA porin (residues 46–159). The protein of interest is fused at the C-terminus of Lpp’OmpA, which will be displayed on the outer leaflet in the outer membrane.
Figure 1. Schematic view of the Lpp’OmpA system
Usage and Biology
We attach the enterokinase site-small peptides-GST tag to the C-terminus of Lpp’OmpA. Once displayed on the outer leaflet, the DDDDK enterokinase cleavage site can be recognized by enterokinase and small peptides will be cut off, which will be detected in the extracellular environment.
Figure 2. Schematic view of the part, taking the intestinal environment as an example
Design
The key component, Lpp’OmpA, refers to the literature by Nicchi, S et, al(2021). Its codon is optimized for E.coli. It contains the T7 promoter and the lac operator to initiate the induced display in the proof-of-concept. The enterokinase site, small peptides QEP, and GST tag were fused at the C-terminus of the Lpp’OmpA. Also, the double terminator is designed to terminate the transcription.
Figure 3. Design of T7-lac-lpp’ompA(codon optimized)-DDDDK-QEP-GST-dt
Experiments
This part is the key element we use in the proof-of-concept of the secretion system, and it helps us better understand the secretion event. Also, it is used to measure the basic parameters of the secretion event. We inserted it into pET-28a plasmid and transformed it into Escherichia col BL21.
The overall experiments we conducted on BBa_K4630100 include:
- Verification of the transformation
- Verification of the protein expression
- Verification of the surface display
- Verification of the enzymatic cleavage
The protocol can be checked in our wiki’s experiment page:
Results
Strain Construction
We successfully transformed our working plasmid into Escherichia coli BL21 (fig 4, fig 5).
Fig 4 Gel electrophoresis results of PCR products from transformed bacterial colonies
Fig 5 Sequencing result of the working strain
Protein expression verification
To confirm the expression of Lpp'OmpA-peptides in bacteria from the transformed plasmids, we performed Western blot experiments. We observed that induction temperature affects the Lpp'OmpA-peptide expression under the T7-lac promoter. We also observed that T7-lac promoter was leaking.(fig 6)
fig 4. The Western blot result for AQ_GST
Surface display verification
Immunofluorescence assay
To assess the surface exposure of the chimeras, we performed fluorescence microscopy on the working strain. The working strain was induced overnight at 16°C. However, under this condition, our negative control also showed a positive result. The negative control will display a lipoprotein on the inner leaflet of the outer membrane. Bacteria were incubated first with corresponding Mouse anti-tag antibodies. Subsequently, samples were incubated with the ABflo® 594-conjugated Goat anti-Mouse IgG (H+L). The ABflo® 594 can be visualized in red, the DNA in blue (DAPI), and the membranes in green (Oregon green).（fig 7）
Fig 7 Immunofluorescence assay for AQ_GST and Negative control
FACS
The surface exposure of all constructs was also verified by FACS analysis. Bacteria were incubated first with corresponding Mouse anti-tag antibodies Subsequently, samples were incubated with the FITC-conjugated Goat anti-Mouse IgG (H+L). This time we found similar results: not only QEP_GST had positive results, but also the negative control.(Fig 8)
Fig 8 FACS analysis for AQ_GST
L1: lptE_FLAG, a strain that will display lipoprotein_FLAG to the outer leaflet of the outer membrane. L1_GST denotes the L1 incubated with GST primary antibody as a control to prevent the unspecific binding. N1:Negative control; AG: AQ_GST
Cryo-EM
To observe the structure of bacteria more clearly, we performed cryo-electron microscopy. Bacteria were incubated first with corresponding Mouse anti-tag antibodies Subsequently, samples were incubated with the 12nm Colloidal Gold-AffiniPure™ Goat Anti-Mouse lgG (H+L). Similar results were obtained. Both negative control and chimeras showed positive results.(fig 9)
Fig 9 Cryo-EM experiment for AQ_GST
Enzymatic cleavage experiment
To confirm that the enterokinase can target the DDDDK site and successfully cleave off the peptide into the environment, a series of enterokinase digestion experiments and western bolt experiments are conducted. We first designed experiments to investigate the minimum digestion concentration of enterokinase, digestion temperature, pH, and duration. Then, we expressed AQ-GST, QEP-GST, and lptE-GST under the environmental factors that we explored before. There are six possible circumstances we found in the following experiments: (1) bacteria membranes are intact and the proteins are presented on the surface, (2) bacteria membranes are not intact (bacteria are broken during induction or digestion), (3) bacteria membranes are intact and the proteins are not present on the surface, and the mixture of (1) and (2), (2) and (3), or (1) and (3) (Fig. 10). The bacteria culture was centrifugated after induction and digestion. The supernatant and precipitation are incubated with two different antibodies. To get a clearer picture, the hypothesized western blot results of different circumstances are demonstrated below (Fig. 11).
Fig. 10 The schematics of deductions of the first three circumstances. 1) bacteria membranes are intact and the proteins are presented on the surface 2) bacteria membranes are not intact. (bacteria are broken during induction or digestion) 3) bacteria membrane are intact and the proteins are not present on the surface.
Fig. 11 The deduction of western blot results of supernatant and precipitation incubated with two different antibodies. 1) precipitation incubated with anti-FLAG antibody 2) precipitation incubated with anti-GST antibody 3) supernatant incubated with anti-GST antibody 4) supernatant incubated with anti-FLAG antibody
Phase 1: the exploration of digestion factors of enterokinase To investigate the environmental factors affecting digestion—specifically the minimum enterokinase concentration, digestion temperature, time, and pH—we conducted a series of experiments. In each experiment, subsequent tests were adjusted based on the optimal conditions identified in the previous stage. By progressively aligning the experimental conditions with those of the intestinal environment, successful in vitro digestion may suggest similar effectiveness in vivo.
We tested the effect of digestion temperature (25°C and 37°C) and digestion time (3 and 5 hours) using 2U of enterokinase in the same buffer (Fig. 12). In our exploration of optimal digestion temperature and time, we induced bacterial expression at 16°C overnight and at 37°C for 1 hour using 1mM IPTG, as bacterial lysis was observed in previous experiments. We hypothesized that the lysis was caused either by prolonged induction at low temperatures or by extended digestion in the nutrient-deprived buffer. In the precipitation-FLAG sample, no signal was observed at 15 kDa in plus enzyme group, corresponding to Lpp’OmpA-FLAG. Additionally, the absence of degraded proteins in the precipitation-GST and supernatant-FLAG samples suggests no bacterial lysis occurred. In the supernatant-GST sample, a comparison of plus and no enzyme group at 37°C for 1 hour showed successful digestion of Lpp’OmpA-FLAG-QEP-GST. However, the presence of degraded protein in the supernatant-GST sample from the 16°C, 12-hour induction with negative enzyme concentration supports the hypothesis that bacterial lysis was caused by prolonged low-temperature induction. From the results, we can tell that the group with 37℃-1h induction and 25℃-5h and 37℃-3h digestion can not only keep the bacteria intact but also suceessfully digest chimeras as the AQ-GST signals are positive in 2 groups.
Fig. 12 Digestion of Lpp’OmpA-FLAG-AQ-GST to explore digestion temperature and time. Bacteria is induced by 1mM IPTG under 16℃ 12h and 37℃ 1h.
Following that, we varied the pH of the 25 mM Tris-HCl buffer to 6.5, 7.5, and 8.0 (the pH range of the intestine) and digested the mixture at 37°C for 3 hours with 2U of enterokinase (Fig. 13).
In our exploration of optimal digestion pH, we induced bacterial expression at 16°C overnight and at 37°C for 3 hours using 1mM IPTG, as the 37℃ 1h induction signal is quite weak. We hypothesized that the weak signal is due to the incomplete presentation of chimeras on the outer membrane. In the precipitation-FLAG sample, no signal was observed at 15 kDa in positive enzyme group, corresponding to Lpp’OmpA-FLAG. Additionally, the absence of degraded proteins in the precipitation-FLAG and supernatant-GST samples suggests bacterial lysis occurred. Although the results of the supernatant-GST in positive enzyme group in both induction group showed the successful digestion of Lpp’OmpA-FLAG-QEP-GST, the digestion occurring in the lysed bacteria in both groups did not confirm that the process was targeted to the bacterial surface. However, the presence of weak signals in the supernatant-GST sample from the 37°C, 3-hour induction does not support the hypothesis that the weak signal is due to incomplete presentation because of short induction time. From the results of group with 37℃-3h induction and pH 7.5 digestion and group with 16℃-12h induction and pH 6.5 digestion shows that positive pH digestion range can cover the pH range of intestine environment.
Fig. 13 Digestion of Lpp’OmpA-FLAG-AQ-GST to explore digestion temperature and time. Bacteria is induced by 1mM IPTG under 16℃ 12h and 37℃ 3h.
Phase 2: expression of AQ-GST under optimal digestion conditions
For AQ-GST, no degradation protein signal indicates no bacterial lysis. In supernatant-GST, with negative enzyme concentration, the Lpp’OmpA-FLAG-AQ-GST signal also indicates the incomplete digestion(Fig. 14).
Fig. 14 Digestion of Lpp’OmpA-FLAG-AQ-GST, Lpp’OmpA-FLAG-QEP-GST and Lpp’OmpA-FLAG-lptE-GST. Bacteria is induced by 1mM IPTG under 37℃ 3h.
Discussion: We also conducted an experiment to investigate whether there will be some bacteria resuspended in the supernatant, which may cause the same results as bacterial lysis. In this experiment, there is no difference in filter used and no filter used sample preparation. It may mean that there is no bacteria in the supernatant without the filter(Fig. 15).
Fig. 15 Digestion of Lpp’OmpA-FLAG-AQ-GST with or without filter used. Bacteria is induced by 1mM IPTG under 37℃ 3h.
Reference
1.Nicchi, S., Giuliani, M., Giusti, F. et al. Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems. Microb Cell Fact 20, 33 (2021).
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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