Team:SZPT-CHINA/Results

Results

Exploratory experiments

Antipseudomonal drug production module

C-di-GMP signaling and BC film production module

Safety and drug release module

● Exploratory experiments
1. MIC values
Firstly, we tested the minimal inhibitory concentrations (MIC) of 4 antibiotics against growth of Gluconacetobacter hansenii ATCC 53582, and the result was detailed in Table 1. The MIC data here would be employed in the following experiments.
Number Antibiotics MIC(ng/mL)
1 Gentamicin 300
2 Kanamycin 180
3 Chloramphenicol 148
4 Ampicillin 100
Table 1. MIC values of different antibiotics against G. hansenii ATCC 53582
2. Culture exploratory experiment
To identify the optimal culture medium for G. hansenii ATCC 53582, an array of growth media was selected for testing the bacterial growth. HS culture medium is commonly used for G. hansenii ATCC 53582, while HS-6, HS-7, and HS-8 are also utilized in some previous studies. Additionally, due to the similarity of medium composition to HS (Table 2), LB and SOC medium were included in our assay here. Altogether, 6 different bacterial growth media were employed to culture the wild type G. hansenii ATCC 53582 and J23100-mcherry-pSEVA331-G. hansenii ATCC 53582.
Medium Formula
HS 2% glucose,0.5% peptone,0.5% Yeast Extract ,0.68% NaHPO4·12H2O,0.15% Citric Acid Monohydrate
LB LB Broth
SOC SOC Broth
HS-6 8% glucose,1% peptone,0.2% Citric Acid Monohydrate,0.27% NaHPO4·12H2O,0.03% MgSO4·7H2O
HS-7 8% glucose,2% YeastExtract,0.2% NaHPO4,0.1% 12H2OKH2PO4,0.02% MgSO47H2O 1% C2H5OH
HS-8 2% sucrose, 0.5% peptone,0.5% Yeast Extract, 0.68% NaHP04·12H2O,0.15% Citric Acid Monohydrate
Table 2. Formula of 6 different bacterial growth media used in our assay
Consistent with visual observation, the optical density of bacteria grown in HS, SOC and HS-8 indicated bacterial growth, whereas bacteria in other media failed to grow, as illustrated by low or even negative values of OD600. In comparison, following 48 hours of culture, a higher level of biomass for bacteria grown in SOC, HS, LB and HS-8 was observed, with the highest OD600 observed in SOC medium. Thus, SOC might be the optimal growth medium. Notably, OD600 of bacterial culture in HS-8 at 48 hours was lower than that in 24 hours, an indicative of transition into the death phase in HS-8 at this timing. Taken together, based on the growth assay using a range of culture media, SOC and HS were opted for the further experiments concerning G. hansenii ATCC 53582.
Figure 1. (a) Liquid culture of J23100-mcherry-pSEVA331-G. hansenii ATCC 53582 after 24 hours of agitation, (b) liquid culture of the wild type G. hansenii ATCC 53582 after 24 hours of agitation, (c) optical density of J23100-mcherry-pSEVA331-G. hansenii ATCC 53582 and wild type G. hansenii ATCC 53582 in different culture media after 24 hours of agitation, (d) liquid culture of J23100-mcherry-pSEVA331-G. hansenii ATCC 53582 after 48 hours of agitation, (e) liquid culture of G. hansenii ATCC 53582 after 48 hours of agitation, (f) optical density of J23100-mcherry-pSEVA331-G. hansenii ATCC 53582 and wild type G. hansenii ATCC 53582 in different culture media after 48 hours of agitation.
To quantify the bacterial cellulose (BC) production of G. hansenii ATCC 53582 in SOC and HS medium, we launched liquid culture of J23100-mcherry-pSEVA331-G. hansenii ATCC 53582 in HS and SOC medium with different volumes (2mL, 2.5mL and 3mL) for 4 days. Then we measured the dry weight of the BC film, which was consistent with the BC thickness as visually observed (Figure 2. a, b). Intriguingly, bacteria cultured in 3mL of HS medium yielded the highest amount of BC, even though the OD600 of bacterial culture in HS was lower than that in SOC. In the end, we chose HS medium to culture and product BC film.
Figure 2. (a) Visualization of bacterial cellulose production in 2mL, 2.5mL and 3mL of HS and SOC medium after 4 days of fermentation in 12-well plates, (b) Quantification of the bacterial cellulose dry weight.
3. Eletrotransformation condition exploratory experiment
In order to optimize the transformation efficiency of plasmid into G. hansenii ATCC 53582, we conducted electroporation assay with the different voltage values, either 2.5kV or 3kV. A larger amount of bacterial transformants can be found on selective plates in the 3kV-treated group. Therefore, we chose 3kV to perform the electroporation of G. hansenii ATCC 53582 with plasmids in the following experiment.
4. Illumination intensity exploratory experiment
During the consult to the experts( For more detail, please click Human_Practices), suggestions were made to explore the effect of details intensity on G. hansenii ATCC 53582 product BC in the NIR light intensity in the c-di-GMP signaling and BC film production module. Then we cultured respectively J23100-bphS-J23109-fcsR-pSEVA331-G.hansenii ATCC 53582 under 5， 10， 20， 50μW/cm2. The yield under 5, 10, 20, 50 μW/cm2 illumination intensity had no significant difference. Therefore, we still chose 50μW/cm2 for film production experiment.
Figure 3. The BC yield at different illumination intensity
● Antipseudomonal drug production module
1. Goal
The function of this module is to produce a chimeric bacteriocin that can target Pseudomonas aeruginosa while having a broader antipseudomonal spectrum than pyocin S2. To enhance the antipseudomonal effect, we further engineered the RBS strength to modulate the expression of SE protein.
2. What work we have done
① Verify that the SE fusion protein has an antibacterial effect on P. aeruginosa.
② Successfully construct a series of plasmids to express different levels of SE protein.
③ Through the inhibition zone test and growth curve test, screen G. hansenii ATCC 53582-derived strains with a pronounced antipseudomonal effect.
3. Proof
3.1 Antipseudomonal properties of SE protein
IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein expression in E. coli BL21 with S2-PET28A or SE-PET28A at 25℃ for 12 hours. Supernatant containing SE or S2 proteins were obtained by sonication and centrifugation. The crude protein extract was loaded into SDS-PAGE gel and stained by Coomassie Blue to verify the presence of target proteins ( For more detailed steps, please click Experiments). After IPTG induction, S2-PET28A-BL21 produced a 83.9 kDa protein (lane 2) while SE-PET28A-BL21 produced a 81 kDa protein (lane 3). In contrast, non-induced E. coli BL21 cells did not express S2 or SE protein (lane 1, lane 4). The results showed that the constructed plasmids S2-PET28A and SE-PET28A could be used to express the fusion protein (Figure 4. a).

Then, supernatant containing SE or S2 protein was respectively added into the culture of Pseudomonas aeruginosa PAO1, P. aeruginosa PAO1 Δ1150-1151（PAO1 Δ1150-1151）and E. coli MG1655. PBS was used as a negative control. Inhibitory effect of supernatant on the P. aeruginosa growth was determined by measuring OD600 using SYNERGY H1 micro-plate reader. The result showed that the SE-fusion protein could prevent P. aeruginosa growth, irrespective of the presence of the PA1150-1151 region, while pyocin S2 can only inhibit the PAO1 Δ1150-1151 mutant, but not the wild type strain. In addition, both SE and S2 proteins had no effect on E. coli growth (Figure 4. b).
Figure 4. (a) SDS-PAGE analysis of protein expression in engineered E. coli. Lane 1 S2-PET28A BL21 without IPTG induction, lane 2 supernatant of S2-PET28A BL21 with induction by 0.1mmoL/mL IPTG, lane 3 supernatant of SE-PET28A BL21 with induction by 0.1mmoL/mL IPTG, lane 4, SE-PET28A BL21 without IPTG induction, (b) Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and E. coli MG1655 treated by the supernatant of S2-PET28A-BL21 or SE-PET28A-BL21 lysate for 790 minutes. PBS was used as the negative control.
3.2 Screen and identify an RBS to express SE protein with an optimal antipseudomonal effect.
Using high-throughput screening, we engineered the strength of RBS that control the SE protein expression level in E. coli, and the resultant plasmids were extracted and in turn introduced into the G. hansenii ATCC 53582. G. hansenii ATCC 53582 culture supernatant containing SE proteins that were expressed under control of different RBS were obtained by sonication and centrifugation and further purified by infiltration with micro-pored membrane. Then, supernatant in different groups was individually mixed with PAO1 culture, and incubated at 30℃ for 12 hours. SYNERGY H1 micro-plate reader was used to measure the OD600 to determine the effect on PAO1 growth. As shown in Figure 5 a, supernatant of culture derived from the 3rd, 4th, 7th, 8th colony had significant antipseudomal effect.

3 μL of the supernatant containing SE protein was dropped on FAB plate with PAO1 evenly spread (for more detailed steps, please click Experiments). Then, the plates were incubated at 30℃ for 12 hours. Supernatants from all the G. hansenii isolates have formed inhibition zone, yet with different sizes. Isolate 3rd and 4th displayed the most significant antipseudomonal effect. The results of the optical density measurements of PAO1 growth and the inhibition zone prove that SE protein is successfully expressed in the engineered G. hansenii. A series of G. hansenii containing the SE expression module with difference RBS were constructed to screen for the strain with the best antipseudomonal property. As shown in Figure 5, the 4th strain pR-RBS300-SE-J23118-IMM-pSEVA331-G. hansenii ATCC 53582-4# is best as it has the largest inhibition zone area.
Figure 5. (a) Optical density of PAO1 treated with the supernatants of different bacterial strain lysates cultured after 12 hours of growth in PBS, (b) Drop plate assay to check the inhibitory effect of SE proteins on Pseudomonas aeruginosa PAO1, (c) Strains used in Figure (b).
● C-di-GMP signaling and BC film production module
1. Goal
As bacterial cellulose (BC) production in G. hansenii is regulated by the second messenger c-di-GMP. Therefore, we aimed control BC film production by regulating c-di-GMP concentration, i.e. so that G. hansenii ATCC 53582 can produce BC under illumination of Near Infrared light (NIR light) at 680nm, but cannot under dark conditions.
2. What work we have done
① Construct the synthesis and hydrolysis modules of c-di-GMP in G. hansenii ATCC 53582.
② Screen the best modules for BC film production, and integrate them into one single plasmid.
3. Proof
We used bphS which is the coding part of the photo-activated diguanylate cyclase BphS as well as the coding parts of c-di-GMP phosphodiesterase from different bacteria to construct the plasmids for c-di-GMP synthesis module and c-di-GMP hydrolysis module in E. coli competent cells. Then the plasmids were extracted and transferred into the G. hansenii ATCC 53582.
Figure 6. (1) The genetic circuit for c-di-GMP synthesis module, (2) The genetic circuit for c-di-GMP hydrolysis module B, amplification of different regions by PCR, (c) Plasmids used in Figure B.
According to the 12-well plate test (Figure 7. A, for more detailed steps, please click Experiments), BC was dried and weighted. A marked difference in BC production between J23100-fcsR-pSEVA331-G. hansenii ATCC 53582 and our control group pSEVA331-G. hansenii ATCC 53582 was observed (Figure 7. B1). Therefore, J23100-fcsR-pSEVA331 was shown to have a marked hydrolytic function. On the other hand, J23100-bphS-bphO-pSEVA331-G. hansenii ATCC 53582 produced a higher level of BC under the illumination of NIR light than the dark condition (Figure 7. B2). Based on the above results, we chose BphS and FcsR as the c-di-GMP synthetase and hydrolase respectively in our system.
Figure 7. A. Verification experiment of BC film yield in 12-well plates B. (1) BC yield by J23100-fcsR-pSEVA331-G. hansenii ATCC 53582, J23100-yhjH-pSEVA331-G. hansenii ATCC 53582, J23100-rocR-pSEVA331-G. hansenii ATCC 53582 and the vehicle control pSEVA331-G. hansenii ATCC 53582, (2) BC yield by J23100-bphS-bphO-pSEVA331-G. hansenii ATCC 53582 under NIR light illumination and dark conditions.
We aim to construct a photo-activated system for production of BC film in G. hansenii ATCC 53582, where the amount of intracellular c-di-GMP (Figure 8. A) sustained at a low level under dark condition, but can reach a high level under illumination of NIR light. To this end, we constructed an array of plasmids by assembling different synthesis and hydrolysis components of c-di-GMP. Finally, these plasmids were introduced into the G. hansenii ATCC 53582 via electroporation to screen the optimal isolate in BC yield (Figure 8. B).

We constructed three plasmids with different promoters that can control the c-di-GMP levels. Then G. hansenii ATCC 53582 were transformed with these plasmids. Then the BC film production in 12-well plates was assessed. Among the strains we tested, a significant difference in the BC yield under different illumination conditions was found for J23100-bphS-bphO-J23109-fcsR-pSEVA331-G. hansenii ATCC 53582 and J23100-bphS-bphO-J23110-fcsR-pSEVA331-G. hansenii ATCC 53582. Notably, a background level of BC production was always detected in all the strains under dark conditions. In the end, we chose J23100-bphS-bphO-J23109-fcsR-pSEVA331-G. hansenii ATCC 53582 which has a significant difference in the BC yield under different illumination conditions as the strain relatively good matches our goal.
Figure 8. (A) The genetic circuits for c-di-GMP signal transduction and BC growth module, (B) PCR identification results, lane 1 J23100-bphS-bphO-J23109-fcsR-pSEVA331-G. hansenii ATCC 53582, lane 2 J23100-bphS-bphO -J23110-fcsR-pSEVA331-G. hansenii ATCC 53582, lane 3 J23100-bphS-bphO-J23119-fcsR-pSEVA331-G. hansenii ATCC 53582, (C) BC film production by different strains, (D) Strains used in Figure C.
● Safety and drug release module
1. Goal
With the illumination of blue light, the expression of the lysis protein controlled by the pDawn promoter would be activated. While the amount of the lysis proteins in the engineered bacteria reaches a certain threshold, the bacteria will be auto-lysed and the antipseudomonal proteins are released, thus antagonizing P. aeruginosa growth.
2. What have we done
① We have identified that the blue light-responsive promoter pDawn can activate the expression of downstream genes in G. hansenii ATCC 53582 under blue light condition.
② We have constructed a set of plasmids which include pDawn to induce the expression of an antiholin-free Lambda lysis cassette (S105), φX174 phage lysis gene (X174 E), and LKD16 lysis cassette (LKD16). After introducing these plasmids into E. coli, bacterial strains grow normally under dark conditions but do not under 470nm blue light.
③ The plasmids will be extracted from E. coli and subsequently introduced into the G. hansenii ATCC 53582, where the expression of X174 E can be activated under illumination of blue light
3. Proof
3.1 Functional verification of pDawn promoter in response to blue light in G. hansenii ATCC 53582.
The plasmid pDawn-RFP-pSEVA331 was firstly constructed and introduced in G. hansenii ATCC 53582, where its function was tested. To be specific, bacteria were cultured under either the blue light at 470nm or dark conditions for 24 hours, and the intensity of red fluorescent signal was detected using fluorescence microscope. After 24 hours of growth, G. hansenii ATCC 53582 in the blue light treated group showed red fluorescence, but no fluoresces were observed for those under dark conditions (Figure 9). Based on these results, pDawn in G. hansenii ATCC 53582 can be successfully activated by blue light irradiation.
Figure 9. Red fluorescence photo of the pDawn-RFP-pSEVA331-G. hansenii ATCC 53582 cultured under 470-nm blue light or dark conditions for 24 hours.
3.2 Functional verification of lysis protein in G. hansenii ATCC 53582
We employed the blue light-responsive promoter pDawn to control the expression of different lysis genes, including S105, X174 E and LKD16 (Figure 10). Random primer-guided mutagenesis on the target RBS position was performed (BBa_B0034) ( For more detailed steps, please click Improvement). To verify the lysis effect of these components, we conducted the plate dot assay ( For more detailed steps, please click Experiments). Expression of lysis genes will be considered effective when bacterial colonies can grow normally in the dark, but not under blue light irradiation.
Figure 10. Genetic circuit for safety and drug release module.
To be specific, 20 single colonies on the culture plate were picked to spot on two parallel plates (if the number of single colonies on the culture plate is less than 20, picked all of them). The plates were incubated either in the dark or under blue light illumination for 24 hours to observe the colony morphology, based on which we determine whether there is a lysis effect.

Although the colony of S105 could not grow under blue light its growth state was abnormal in the dark. We attributed this phenomenon to a basal expression level of lysis protein controlled by pDawn promoter (Figure 11. A (a1), (a2)). Of note, the 4th isolate of with pDawn-X174 E-pSEVA331- E. coli grew normally in the dark but not under the blue light irradiation（Figure 11. A (b1) and (b2)）. Additionally, as shown in Figure 11. A (c1) and (c2), all the colonies of E. coli with pDawn-LKD16-pSEVA331- E. coli were able to grow under both conditions, whereas the growth of 4th, 9th,10th and 12th colonies was remarkably inferior than those in the dark, indicating that pDawn-LKD16 allowed bacteria to be lysed in response to blue light, yet with a modest effect.

In order to construct G. hansenii ATCC 53582 strains with the capability of light-responsive cell lysis, we firstly introduced the plasmids that have lysis effect in E. coli into the G. hansenii ATCC 53582, and then 8 single colonies were picked to perform the drop plate assay. Two copies of plates were separately incubated under the blue light and dark conditions at 30℃. The bacterial colony morphology, including the size and thickness, was observed every 24 hours to determine the lysis effect. After introducing the plasmid containing S105, X174 E or LKD16 into the G. hansenii ATCC 53582, they can cause similar colony-lysis phenotype. While the plasmids containing pDawn-S105 completely inhibited growth of G. hansenii ATCC 53582 under both conditions. (Figure 11. B (a), (b) and (c)). We thus preserved the pDawn-X174 E-pSEVA331-G. hansenii ATCC 53582 and pDawn-LKD16- pSEVA331-G. hansenii ATCC 53582 . And these strains were spotted on the culture plates for screening. The strain pDawn-X174 E-pSEVA331-G. hansenii ATCC 53582-7# showed a stable lysis effect, while the others did not. (Figure 11. C). In the end, we chose pDawn-X174 E-pSEVA331-G. hansenii ATCC 53582-7# which can be activated under illumination of blue light as the goal strain.
Figure 11. Drop plate assay to assess the inducible production the lysis protein by 470-nm blue light illumination, (A) E. coli DH5α transformed with different lysis proteins, (B) G. hansenii ATCC 53582 transformed with different lysis proteins, (C) pDawn-X174 E-pSEVA331-G. hansenii ATCC 53582, which stably showed the lysis effect, (a1) and (a2), pDawn-S105-pSEVA331, (b1) and (b2), pDawn-X174 E-pSEVA331, (C1) and (C2), pDawn-LKD16-pSEVA331,(a1), (b1) and (C1) are incubated under 470-nm blue light, (a2), (b2) and (C2) are incubated under dark conditions.