Construction of NANO-AI-Cleaner
Overview:
we have already successfully constructed the plasmid (BBa K3712015) and engineered an intelligent temperature-controlled and user-friendly expression system to express two small drug molecules BLP-7 and TLR2 antagonist with ELP to achieve in situ response at the lesion ,verifying the feasibility of four molecules expression Wild-type BLP-7-27 ELP (BBa K3712023), Optimized BLP-7-27 ELP (BBa K3712018), Wild-type TLR2 antagonist-27ELP (BBa K3712022), and Optimized TLR2 antagonist-27 ELP (BBa K3712008).
Methods:
After GenScript synthesized the gene fragments, we use in-fusion cloning method to construct them in the pVB plasmid respectively, which is transformed into E. coli Nissle 1917, then culture the strains at 37\(^\circ\mathrm C\) for 12 hours, and induce at 45\(^\circ\mathrm C\) for one day. Then, SDS-PAGE is performed to prove the successful expression by molecular weight, and Western Blot experiment is conducted to confirm the expression results.
Results:
The gene fragments of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, Optimized TLR2-27 ELP were synthesized at GenScript, and was constructed in the pVB plasmid (Figure1).
Figure 1: The agarose gel electrophoresis map of Wild-type BLP-7-27 ELP (BBa K3712023), Optimized BLP-7-27 ELP (BBa K3712018), Wild-type TLR2 antagonist-27ELP (BBa K3712022), and Optimized TLR2 antagonist-27 ELP (BBa K3712008) respectively constructed in pR/pL expression system. About 200-1000 ng of plasmid was digested at 37 degree centigrade for 30-60 minutes and analyzed on 1% Agarose Gel. Lane1: circular plasmid (brighter) and supercoiled structure; Lane2: plasmid (brighter) digested by EcoRI and BamHI and target gene fragment.
Then we transform the constructed plasmids into E. coli Nissle 1917 to express the target proteins. All transformed E. coli Nissle1917 are cultured at 37\(^\circ\mathrm C\) for 12 hours and induced at 45\(^\circ\mathrm C\) for one day. The bacterial that were only cultured at 37\(^\circ\mathrm C\) for 12 hours without transformation and the bacterial transformed without induction were used as the control group. The experimental group is induced at 45\(^\circ\mathrm C\) for one day after transformation and culturation. The standard curve was made by BCA assay. By determinating the appropriate loading concentration, the loading amount of each channel was controlled to 30\(\mu\)g, and then perform SDS-PAGE to verify the expression of our product.
Figure 2: SDS-PAGE of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2 antagonist-27ELP, Optimized TLR2 antagonist-27 ELP expression results. Near the 25kDa marker, the binds of Wild-type TLR2-27ELP and Optimized TLR2-27 ELP after induction are darker than the expression system without induction. Near the 15kDa marker, the bands of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP after induction are darker than the expression system without induction, which indicates that our constructed pR/pL temperature control system is able to express our product in E. coli Nissle1917.
Western Blot is conducted to further verify that the molecule near the marker is the right target protein we want to express. After transmembrane, we incubate overnight with the primary antibody against His-tag, and then the secondary antibody for development. After induction, there are obvious bands, as shown in Figure 3, which further proves the success of vector construction and expression of our target protein.
Figure 3: Western blot results of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP. A) In the vicinity of 25kDa, the color of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction have obvious color differences. B) Around 25kDa, the color development of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction is different obviously.
Future direction:
(1) Due to time and guidance issues, we could not make up the Western blot with internal parameters, but we can guarantee it is our target protein. Because after the transformation was successful, we stained with Ponceau red to observe obvious bands. Later, we will complete the Western blot with internal parameters as a control to further illustrate the expression of the four proteins Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP level.
(2) In order to better verify the aggregation properties of ELP, the bactericidal properties of antimicrobial peptides and the anti-inflammatory effects of antagonists, we will continue to express the products we need in the future to further verify the functions of the proteins we need.
Purification
Overview:
After clarifying that the above four kinds of proteins successfully expressed in E. coli Nissle 1917, we successfully purified a large amount of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP. Unfortunately, we have not been able to purify Wild-type TLR2-27ELP and Optimized TLR2-27 ELP. The results of SDS-PAGE only show the molecular weight of 27ELP. Therefore, we speculated that passing through the nickel column caused a break between Wild-type/Optimized TLR2 antagonist and 27ELP.
Methods:
The above-mentioned strains that can successfully express products are cultured in LB medium at 37\(^\circ\mathrm C\) for 12 hours, and induced at 45\(^\circ\mathrm C\) for 24 hours. Then the cultures are centrifuged at 6000 rpm to collect the bacteria that are suspended and concentrated with PBS. After that, add lysozyme at 1:100 and PMSF at 1:1000 when lysing on ice for 30 minutes, and then disrupt ultrasonically at 400W. Centrifuge the turbid liquid again to collect the supernatant, filter and sterilize, and pass the sterilized liquid through FPLC for nickel column affinity chromatography. Then we use BCA to measure the concentration of the product, and perform SDS-PAGE for product conformation.
Results:
After culturing, induction and sterilization, the results of nickel column affinity chromatography show that both of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP elution have single peaks at 80 mM with good symmetry, which preliminarily indicates that the target protein is purified. Then we take the BCA method to determine the concentration (Table1) and SDS-PAGE to verify the existence of product (Figure4).
| sample | concentration |
|---|---|
| Wild-type BLP-7-27 ELP | 1.5mg/ml |
| Optimized BLP-7-27 ELP | 1.2mg/ml |
Figure 4: The elution peak height of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP and the running gel spectrum after purification. A) The elution peak height of Wild-type BLP-7-27 ELP is 1.41; B) The height of the Optimized BLP-7-27 ELP elution peak is 1.26; C) SDS-PAGE, lane 1/2 is two-tube stream, lane 3 For Wild-type BLP-7-27 ELP, a thicker band appears near the 15kDa marker; lane 4/5/6 is a three-pipe stream that collects Optimized BLP-7-27 ELP, and a thicker band appears near the 15kDa marker.
Future direction:
(1) Unfortunately, due to time issues, we have no way to complete the purification of TLR2 antagonist. In the future, we will try different linkers to connect Wild-type/Optimized TLR2 antagonist and 27ELP.
(2) Although our purified samples will not affect the subsequent functional verification, it can be seen from SDS-PAGE that even the purified bands will also have miscellaneous bands. Therefore, we want to use molecular sieves and freeze dryers for further purification, attempting to obtain purer samples and make our experiment more successful.
Functional testing
ELP
Overview:
We use Wild-type BLP-7-27 ELP to verify the aggregation function of ELP, because we want to measure the aggregation ability if linked with another protein, but another protein can't be too large to guarantee the aggregation of ELP, so we use the ELP connected with the Wild-type BLP-7 for ELP functional verification.
We first verify that the molecular size of ELP has changed under the heating condition of 30~50\(^\circ\mathrm C\) with dynamic light scattering. At the same time, we also measure the change of zeta potential of the ELP during the aggregation process. An inflection point appears at 44\(^\circ\mathrm C\), which preliminarily indicates that the ELP aggregates at 44\(^\circ\mathrm C\). Furthermore, we use transmission electron microscopy and atomic force microscope. We have observed an ellipsoid structure. This is because the energy obtained by the ELP in the aggregation process is not complete and the Wild-type BLP-7 connected to the end of the ELP. As a result, ELP only assemblies into a spherical shape and it is difficult to form a perfect spherical structure.
However, this still fulfills our requirement for temperature-controlled aggregation, skin repair, and increasing drug concentration at lesion and prolonging administration time.
Methods:
We use Wild-type BLP-7-27 ELP (1.5mg/ml) to measure its aggregation ability by dynamic light scattering. Under the condition of 30~50\(^\circ\mathrm C\), equilibrate for 5 minutes and increase the temperature by 1\(^\circ\mathrm C\) to obtain continuous change of the size and zeta potential of ELP aggregation under this temperature gradient. Then, we use the same batch of samples to incubate at a constant temperature of 44\(^\circ\mathrm C\) for 20 minutes. Then we use transmission electron microscope and atomic force microscope to observe the morphology.
Results:
Under the temperature gradient of 30~50\(^\circ\mathrm C\), the particle size measurement is carried out for every 1\(^\circ\mathrm C\) increase. We find that the main peak of ELP aggregates distribution (more than 70%) gradually shifted to the larger particle size as the temperature increased. Location gathering. We averaged the main peaks of the three particle diameter measurements, and then plotted the average value as shown in Figure 5.A. We found that there is an inflection point at 44\(^\circ\mathrm C\), which means that 44\(^\circ\mathrm C\) is the transition temperature of Wild-type BLP-7-27 ELP. At the same time, we found that at 44\(^\circ\mathrm C\) there is a rising inflection point for the Zeta potential, which is caused by rich positive charges of the Wild-type BLP-7 connected with the ELP aggregate.
Figure 5: Changes in particle size and zeta potential during ELP aggregation. A) The particle size of the ELP aggregate changes. The particle size is around 10nm before 44 degree centigrade, then it reaches 100nm or more at 44 degree centigrade, and thereafter it rises sharply. B) Changes in zeta potential of ELP aggregates. The change is relatively flat before 40 degree centigrade, and the value is around -5mV; at 40 degree centigrade, there is a sharp drop. That's because ELP has accumulated locally, but no aggregates are formed. The positive charge of ELP attracts anions in the solution environment, so it appears as a anionic enhancement; the inflection point appears at 43 degree centigrade, where the ELP has been aggregated, and the relative density of the surface charge of the aggregate is lower than the charge density of the dispersed state. Therefore, the absolute value of the charge is reduced, but it will not cause electrical reversal and a particularly drastic change in the amount of charge occur.
We use the same batch of Wild-type BLP-7-27 ELP samples (1.5mg/ml) for the transmission electron microscopy test with the dynamic light scattering test. What stimulates us is that we find there are more spherical structures, but there are some contiguous structures. The reason for these phenomena is that (Figure 6D), these ball-like structures have quantity of positive charges, attracting the negative charges materials around them, and these materials attract positive charges spherical structures again, thus forming some darker adhesions. For the lighter colored adhesions. (Figure 6C) Although the ELP aggregates at 44\(^\circ\mathrm C\), it doesn't completely aggregate. The ELP molecules that do not have enough energy to aggregate a relatively loose adhesion structure with a lower density, so it looks lighter under the transmission electron microscope.
Figure 6: Transmission electron microscope image of ELP aggregates. A) Ball-like structure, the size of ball a is about 500nm, the size of ball b is about 400nm, the scale bar is 50nm, B) the ball-like structure, the size of ball c is about 200nm, the scale bar is 50nm; C) The darker adhesive object is about 2.5 micra, in which the size of the ball-like structure is between 100~300nm, bar 200nm; D) The lighter adhesive object, the whole is about 6 micra, bar 600nm.
After knowing the two-dimensional morphology of the ELP aggregation, we want to see the three-dimensional structure further, so we use the atomic force microscope to observe the surface morphology. What makes us strange is that we have observed many ellipsoids, so we feed back this phenomenon to the modeling team, which gives a good explanation from a mathematical point of view: although the most stable structure is spherical theoretically, but there is not enough energy, so it forms an ellipsoidal structure with incomplete aggregation. (more details here: D value verification and rational interpretation of experimental data.)
Figure 7: Atomic force microscope image of ELP aggregates. The brighter means the higher the depth and the darker the color means the greater the degree of depression, bar means the depth of the height map. A) After incubating at 25 degree centigrade for 20 minutes, there are few ELP aggregates, dispersed. B). Figure A is 3D image of the field of view. C) After incubating at 44 degree centigrade for 20 minutes, there are more ELP aggregates, dense. D) Figure C is 3D image of the field of view. Since the shooting is in a liquid phase environment, the depth is not large and the shape looks relatively flat.
Future direction:
(1) Although the modeling team has given a good verification that 27 ELP has the temperature control effect we expect, in the future we will verify the temperature control aggregation ability of 54 ELP through experiments to further support the ELP aggregation temperature of the modeling group. The applicability of the control aggregation model.
(2) After we purify the Wild-type/optimized TL2 antagonist-27 ELP, we will verify that this product has a similar transition temperature with Wild-type BLP-7-27 ELP within \(\pm\)0.5~1\(^\circ\mathrm C\). (Link: Prediction: ELP with chain length=54)
BLP-7
Overview:
We carry out antibacterial experiments respectively with purified Wild-type BLP-7-27 ELP, optimized BLP-7-27 ELP. Firstly, we build an anaerobic environment for Propionibacterium acnes with Anaerobic bag indicator and hermetic bag and cultured at 180 rpm, 37\(^\circ\mathrm C\). Then we use the inhibition zone method and absorbance method to measure the bacteriostatic ability of the antimicrobial peptides. Unfortunately, due to the preliminary exploration of anaerobic culture and lack of time, we fail to measure the minimum bacteriostatic concentration of the antimicrobial peptides. We will explore the conditions of bacteriostatic experiments in the future.
Methods:
Bacteriostatic experiment one: inhibition zone experiment
Preparing solid medium: Heat and melt the BHI medium, then cool it to about 50\(^\circ\mathrm C\). Add 1 ml of bacterial suspension to every 100 ml medium. Shake to ensure that the bacteria distribute evenly and pour into a large culture dish. Place Oxford-cups on the surface of the plate corresponding to the marked number, and dropwise add antimicrobial peptide sample with concentration gradient to Oxford-cups. Incubate together for a period of time and measure the diameter of inhibition zone.
Bacteriostatic experiment two: absorbance method
Add BLP-7 at concentrations ranging from 1 to 1000\(\mu\)M to the culture medium and incubate anaerobically at 37\(^\circ\mathrm C\) for 24 hours using antibiotics as positive control and sterile water as negative control. The MIC value of BLP-7 is clarified by measuring the absorbance at 630 nm using a microplate reader. Assume the concentration with no significant difference from the positive control group MIC.
Future direction:
(1) Due to the lack of experience and method of Propionibacterium acnes cultivation, we fail to ascertain the optimal growth condition of Propionibacterium acnes after a long period of exploration although we successfully culture it. Subsequent exploration will be carried out to find the appropriate method to control the growth of Propionibacterium acnes.
(2) After determining the optimal growth conditions, we will further explore the principles of antibacterial experiments due to the uncertainty of incubation time and temperature of antimicrobial peptides.
TLR2 antagonist
Overview:
A fracture happens when purifying Wild-type TLR2 antagonist-27ELP and optimizes TLR2 antagonist-27 ELP, thus, we choose to synthesize Wild-type/optimized TLR2 antagonist gene directly and link to pVB plasmid and induce expression at 45\(^\circ\mathrm C\) to confirm the competitive binding ability of TLR2 antagonist to triacylated/diacylated lipoproteins compared to that of TLR2 considering the lack of time to explore the flexibility of linking peptide.
Methods:
Since triacyl-lipoprotein Pam3CSK4 must be dissolved in formic acid (1mg dissolved in 1ml formic acid) and formic acid emits a lot of heat when titrated, we investigate the optimum dilution rate ensuring that the release of heat have no apparent influence on the measure of heat generated from combination between Wild-type TLR2 antagonist-27ELP and optimized TLR2. So we try to titrate in PBS after diluting formic acid 50 times, 100 times, 200 times, 500 times with PBS, and titrate sodium chloride after diluting formic acid 50 times, 100 times, 200 times, 500 times with sodium chloride.
Results:
We find that when formic acid is diluted 100 times with PBS and titrated with PBS, the heat released is minimal and relatively stable. Under this background, the concentration of acylated lipoprotein is 60\(\mu\)M, and the concentration of Wild-type/Optimized TLR2 antagonist is 6\(\mu\)M. Therefore, we are confident of the experimental results.
Figure 8: Heat released in PBS titration with 100 times diluted formic acid. The upper Figure shows the heat released per second at a given point in the titration process. The lower Figure shows the heat released per drop of formic acid diluted 100 times with PBS. Add only 20 microliter formic acid diluted 100 times with PBS for the first drop and 40 microliter for the next 19 drops. At equilibrium, the titration heat released is about 1.50 microcalorie per second and 120kcal per mole of the injected substance.
Future direction:
(1) A purer Wild-type/Optimized TLR2 antagonist should be obtained through molecular sieve and concentrated to conduct ITC experiment.
(2) Dilute PBS with Pam3CSK4 dissolved in pure formic acid for 100 times and titrate Wild-type/Optimized TLR2 antagonist dissolved PBS to prove that optimized TLR2 antagonist has stronger combination ability than Wild-type TLR2 antagonist, which means that optimized TLR2 antagonist has stronger combination ability than TLR2.
Conclusion
Construction of NANOAICleaner:
We successfully construct different expression systems with pR/pL temperature control systems and express Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP and Optimized TLR2-27ELP. The expression products are verified by SDS-PAGE and Western Blot. At the same time, we also verify that the pR/pL temperature control system can work under the condition of infrared irradiation, which provides a good prospect for the application of our system in photodynamic therapy. Based on the solid functional data and modeling analysis, our NANOAICleaner system has specifically smart characteristics for potential biomedical applications.
Purification:
We successfully purify Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP with high concentration and purity, providing good raw materials for subsequent functional verification.
Functional testing:
ELP: We preliminarily prove the aggregation of ELP by measuring the changes of particle size and Zeta potential in the ELP aggregation process by dynamic light scattering, and observe the morphology of ELP aggregates by transmission electron microscopy and atomic force microscopy. Combined with the verification of the molecular modeling, we successfully prove the phase transition temperature and morphological characteristics of ELP aggregates from both aspects of experiment and modeling.
BLP-7: We have figured out a method to culture Propionibacterium acnes, and we are exploring condition to conduct antibacterial experiments.
TLR2 antagonist: We have found out the optimal solution environment for titration, laying a good foundation for the follow-up isothermal titration experiment.