Team:WHU-China/Results

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BC reinforcements BC production Light system CBD detect Streptavidin detect Biotinylation detect DSAOP detect AMP detect

Result

Bacterial Cellulose (BC) Reinforcements

• Microscopic Examination

1.Optical Microscopic Images

The effect of aging and ex-situ reinforcement is clear enough under the optical microscope while in-situ reinforcement is less obvious. Although further observation needs to be done by microscope with higher resolution, it is believed that in-situ reinforcement would not disturb the silk’s texture.

2.SEM (Scanning Electron Microscope) Images

The images vividly indicate the effect of the aging, ex-situ reinforcement and in-situ reinforcement on the silk samples. The images proves that in-situ reinforcement is a more appropriate way to do reinforcement based on the effective aging.

• Functional Test

1. Mechanical strength is improved. In-situ reinforced silk samples is developed in tensile strength to some degree, which stands  for mechanical strength.

2. Softness is improved. In-situ reinforced silk samples become softer than the aged ones because they have larger elongation at break, which helps the silk perform better facing mechanical pressure.

• Chromatic Aberration Analysis

Since the bacteria cellulose is transparent, it is believed that the pattern of the silk would not be affected. Besides, we also managed a color shift experiment to test whether the pattern of the colored silk samples would be affected.

BC Production

We were lucky to get the optimized set of plasmids to produce BC in E. coli from Professor Tomoya Imai and successfully expressed BC in E. coli BL21(DE3) after exploring the conditions for a period of time.

1.Observation with Naked Eyes.

After inducing the expression of CesA, CesB and DGC by IPTG and arabinose for 36 hours, we found flocculent substances were similar to BC in the medium, which made the medium more viscous.

2.In-situ Observation under Scanning Electron Microscope (SEM)

We also sprayed E. coli on the silk and observed with SEM (without cleaning). It is proved that engineered E. coli BL21(DE3) strain has the ability to synthesize and secrete bacterial cellulose, and to reinforce the silk. The secreted BC can attach to and connect between silk fibroins to achieve reinforcement.

3.Quantitative Determination

We also did quantitative determination of BC content by anthrone method after being treated by strong acid. We set bacteria cellulose produced by G.xylinus as positive control, the uninduced E. coli as negative control. By comparing with these two groups, we can confirm that the flocculent substances in the medium are exactly bacteria cellulose produced by E. coli.

Verification of Light System

To realize the irregular reinforcement, we designed a light system to control the E. coli to secret BC in specific site.

1.Light System Validation

1.0-Blue light system

At the beginning of our project, we planned to use the 1.0 light system--blue light system. Thus, we collaborated with ShanghaiTech_China and got a blue light system from them. We tested their system’s feasibility and effectiveness. The results are as follows:

From the comparison of the results between the non-induced group and the induced group, it is clear that more fluorescent proteins can be produced when induced by blue light. There were extremely significant differences. Therefore, we can estimate that the blue light system can achieve the purpose of regulating protein expression. This provided a very reliable light control element for our project.

2.0-Red light system

When we designed the red light system, we considered that the promoter pOmpC may have osmotic noise. Thus we did improvement on it by adding a riboswitch controlled by theophylline. We did experiments to verify that the riboswitch can realize a tight control of this promoter. The following is the results:

What’s more, we did experiments to confirm that the promoter pOmpC with riboswitch can open via theophylline induction under certain concentration.

Thus, the improved pOmpC can be tight controlled as well as be induced effectively. As for the whole system, we constructed complete pathway in E.coli successfully. But time limited, the function verification experiments did not get significant results, we hope we can verify it in the future.

2.Irregular Shaped Controlling

When we got a reliable light system, we can use it to verify our idea of irregular shaped controlling. We created several measurement methods to confirm that idea. Unfortunately, because of the limits of time and experimental conditions, we did not get good results up to now. However, we will try to improve our methods to confirm this idea until the last minute.

Verification of The Cellulose Binding Ability

To verify the cellulose binding ability of the cellulose binding domain, we chose dCBM3a and CBM3a. We set three groups in this experiment, experimental groups of dCBM3a, CBM3a and the negative control--sfGFP. Then the protein samples were mixed with bacteria cellulose homogenate.

The fluorescent residues were measured after incubating the bacterial lysate with BC membrane (pre-dried in a black 96-well plate) and washing for a few times. We confirmed that both of dCBM3a and CBM3a had good cellulose binding ability compared with sfGFP. What’s more, the dCBM3a we improved had higher affinity with bacteria cellulose than CBM3a. (Learn more details Improve)

Streptavidin Detection

Streptavidin can specifically bind to biotin, so we decided to use biotin magnetic beads to detect the existence of streptavidin domain of dCBM3a. After incubating the biotin magnetic beads with engineered bacteria lysate and washing, it was found that the target protein was successfully attached to the biotin magnetic beads by SDS-PAGE detection. We can see that the supernatant after incubation has obviously lower amount of the dCBM3a proteins, while there were a lot of dCBM3a bound to the biotin magnetic beads. Thus, the function of streptavidin domain of dCBM3a had been confirmed to bind to biotin successfully.

Biotinylation Detection

To realize the complete assembly of converter we designed, we had to verify the enzyme BirA can successfully biotinylate target protein with Avi-Tag .

Thus, we co-expressed BirA enzyme and the reporter protein sfGFP with Avi-Tag. By using the method of HABA/Avidin, we detected the biotin level of co-expression system and compared with single sfGFP expression system. We got the results as follows:

According to the results, we know that the sfGFP produced by pBGa are biotinylated well. Thus, we directly proved that the functional peptides with Avi-Tag can be successfully biotinylated via BirA enzyme in vivo in our project. (Learn more details Contribution)

DSAOP Function Detection

To verify the antioxidant ability of DSAOP, we arranged two measurements.

1.Measurement of DPPH Radical Scavenging Activity

The DSAOP antioxidant activity was determined using the DPPH radical scavenging assay.  When free radical scavenger is added to it, DPPH can be combined or replaced to reduce the number of free radicals and make the solution color become lighter. We can use the reduction of absorbance to evaluate the ability of scavenging free radicals. In other words, the antioxidant capacity was calculated by detecting the effect of removing DPPH from the sample at the wavelength of 517nm.The DPPH radical scavenging activity was calculated as follows:

DPPH radical scavenging activity (100%) = [1 − (Ai − Aj )/ A0] × 100.

The following is the result of the DPPH radical scavenging activity of DSAOP compared with DSAOP+Avitag and Vitamin E (positive control).

2.Measurement of Hydroxyl Radical Scavenging Activity

Fenton reaction to generate hydroxyl free radicals react with salicylic acid, generated at 510 nm with special absorption of 2, 3 - dihydroxy benzoic acid. Reaction is as follows: if join in reaction system which has the function of removing hydroxyl free radical was tested, it will reduce the number of generated hydroxyl free radicals. Therefore, it reduce the generation of colored compounds corresponding. Reaction time was fixed. The absorbance of the reaction solution containing the measured substance was measured at 510 nm and compared with the blank solution to determine the hydroxyl content of the measured substance clearance of bases.

The following picture is the result of the Hydroxyl radical scavenging activity compared with DSAOP+Avitag and Vitamin E.

The DSAOP-Avitag has the best hydroxyl scavenging activity and is better than DSAOP. According to this result, we got that adding Avitag may not cause damage on DSAOP’s hydroxyl scavenging activity, but can increase this activity.

AMP Function Detection

The expression and purification of antimicrobial peptides were the difficulties in our project. The expression of such short peptides in E.coli is often prevented by their solubility and effectiveness. At the beginning of the project, we carried out a lot of exploratory experiments, including the direct expression and purification with his-tag, the indirect expression with sumo-tag. Finally, we successfully purified the antimicrobial peptides by using the GST tag system and proved its effectiveness to against mold.(Learn more details Notebook)

The results were as follows:

Firstly, to cleave the GST-tag from the fusion protein, we expressed and purified the Prescission Protease (PPase).

We used the purified PPase to cleave the fusion protein in cleavage buffer hung in 4℃ for 16h. We did the SDS-PAGE electrophoresis.

It was strange that, after cleavage, there was only the protein about 26kDa (may be GST) without the PgD5 in the SDS-PAGE gel. After repeated the experiment many times and analyzed the process, we speculated that we had succeeded in the cleavage but the concentration of PgD5 was too low to be stained by the Coomassie blue. As a result, we did silver staining. It proved our conjecture, so we condensed the cleavage sample by PEG-20000. The SDS-PAGE gel showed that we got the PgD5 successfully!

The next job was to test whether the PgD5 could inhibit the mold. We used the solvent (cleavage buffer in purification, pH~7.0) as the negative control, 10% NaOH as positive control and Aspergillus japonicus as the test object. To test the function of antimicrobial peptides in various pH environments, we adjusted the pH of the PDA medium (pH~5.5) to pH~7.5 and pH~8.0. The results indicated that PgD5 had a strong antifungal effect under pH~7.5 and pH~8.0.