Team:USTC

Proof of concepts

Overview

    our project focused on producing nanoparticles to get through the BBB and establishing an effective bio-manufacturing system to produce this kind of nanoparticle to lower the cost and achieve better therapeutic effect. Our lab work mainly focused on the construction of chassis strains for the production of borneol. Our model had predicted the effect of borneol on improving permeation efficiency, Moreover, our implementation had proved the marketing possibility of our products. It is a rigorous evaluation on the commercial value of the project, indicating the bright future of our product. We will guarantee the reliability of our project based on the feasibility of several stages in wet experience and the core concepts of our model.

Plasmid construction

    When constructing plasmids, we tried plasmid assembly kits from several companies to ensure that the plasmids we designed could be successfully constructed. Based on different assembly principles, we made the following attempts:

a.     Vazyme ClonExpress II One Step Cloning Kit

     Principle based on homologous recombination

b.     Thermo Fisher Fastdigest Restriction Endonuclease and T4 DNA Ligase

     Principle based on restriction enzyme specific site cleavage and end DNA ligase.

c.     In-Fusion® Snap Assembly Master Mix

     Principle based on overlap fragment 5' -3' cut into sticky ends and adhesive ends match each other to be repaired by bacteria

Table1. Three kinds of methods to construct plasmid based on different theory.

Figure 1. Plasmid construction based on homologous recombination. (A) Plasmid assembly based on restriction enzyme and DNA ligase. (B) Plasmid combination based on infusion cloning.

    According to the size of the inserted fragments and the particularity of the sequence, when constructing single-gene plasmids we chose to use ClonExpress II One Step Cloning Kit to assemble plasmids based on the principle of homologous recombination. When constructing the double-gene plasmid, we chose to use Fastdigest Restriction Endonuclease and T4 DNA Ligase to complete the assembly. In the construction of larger plasmids, in consideration of the sequence length of the inserted fragment, we chose In-Fusion® Snap Assembly Master Mix to complete the construction, and achieved satisfactory results. It proves that our plasmids design and assembly are feasible based on different plasmid construction methods.

Exploring the preparation method of competent cell

    At first, we lacked the understanding of the preparation method of Y.lipolytica. Therefore, we conducted a lot of research and asked our PI for help. After thorough investigation, we have obtained the following methods for the preparation of competent cells and were confident in the efficient transformation of Y.lipolytica.

a.     General Preparation Methods of Yeast Competent State (Figure.2)

b.     Preparation of lipolytic yeast electrocompetent

c.     One-step preparation of yeast competent state

    After our testing and comparison, we found that method a had the highest transformation efficiency in the preparation of Y.lipolytica competent cells.

Figure 2. Method c preparation of competent cells and transformation efficiency. (A) Method b preparation of competent cells and transformation efficiency. (B) Method a preparation of competent cells and transformation efficiency. (C)

    However, considering that the protocol is a general method for the preparation of yeast competent cells, we further optimized the protocol according to the particularity of Y.lipolytica, such as adjusting the culture time to achieve the appropriate OD value, increasing the centrifugation speed, etc., and formed a new protocol for the preparation of Y.lipolytica competent cells.

Figure 3. Optimized competent preparation and transformation method for Y.lipolytica.

    This protocol ensures that the plasmids we constructed can be efficiently integrated into the genome of Y.lipolytica.

Exploring the method of yeast colony PCR

    After Y.lipolytica transformation, we plan to determine whether the linearized plasmid is integrated into the genome of Y.lipolytica by yeast colony PCR. Since Y.lipolytica itself also contains the each enzyme gene of MVA pathway. Therefore, when implementing colony PCR, primer design is essential. Considering that we have selected a specific promoter for each enzyme gene which is different from the endogenous promoter, in primer design, the forward primer is located in the promoter of the special sequence and the reserve primer is located in the coding sequence of the enzyme gene, so that we can successfully detect whether the plasmid carrying the enzyme gene successfully transformed.

Figure 4. The theory of colony PCR to test whether the target gene has been transformed to Y.lipolytica.

    But unfortunately, after a number of early attempts, the probability of positive is still ridiculously low. Based on the results, we analyze and find many problems. First of all, Y.lipolytica cells contain complex intracellular components and rich in PCR inhibitors. Considering the detection limit of PCR, we plan to reduce the number of Y.lipolytica in the PCR system to ensure that the DNA polymerase can work properly. In addition, in view of the more complex and thicker cell wall components of Y.lipolytica, we tried a variety of methods for cell wall disruption.

a.     Extend pre-denaturation time in PCR ( 95℃ 10 min )

b.     Extreme cold and hot pretreatment ( -20℃ 5min and then 95℃ 15min )

c.     Quartz sand oscillation crushing treatment

d.     Snail Enzyme digestion Y.lipolytica Cell Wall

e.     Hot alkali digestion wall breaking treatment ( 10mM NaOH 98℃ 15min )

    During our test, we found that method e can get the most positive results in comparison. This effective initial screening method played a crucial role in our selection of the strain, proving that our strain was successfully transformed and the linearized plasmid integrated into the genome.

Figure 5. The first four methods of colony PCR present negative results.

Figure 6. The last method of colony PCR present several positive results.

Determination of production

    In order to obtain our target product borneol from the fermentation environment, we used ethyl acetate to extract the fermentation broth, which ensured that we could successfully separate borneol and determine its yield. Unfortunately, after in-depth investigation, we did not find a suitable chromogenic agent for borneol, which greatly limited our choice of methods for later determination of yield. Luckily relying on the USTC chemical experiment platform, we finally found a reliable and high-resolution detection method Gas Chromatographic Mass Spectrometer ( GCMS ), which confirmed that our chassis strain successfully biosynthesized and produced borneol.

Figure 7. The comfirmation of borneol produced. At 13.23 minutes, the elution peak of suspected borneol appeared. Analysis of mass spectrometry images showed that there was a quantitative ion peak of m / z ≈ 95 in the corresponding time period, which may be related to the presence of borneol.

Feasibility of crossing blood-brain barrier

    Because no further experiments can be carried out, we mainly use modeling to verify the design of this part. First, we calculated the theoretical yield of borneol converted to borneol amine and connected to nhs-peg-PLGA, which is about 73.93% (Figure.7) and meet our expectations. After that, we modeled the diffusion of lipid nanoparticles in the human body and predicted the proportion of lipid nanoparticles passing through the blood-brain barrier based on the classical atrioventricular model (Figure.8). Through the simulation of the model, we can draw that about 3.8385×10^(-3) g/L of lipid nanoparticles can pass through the blood-brain barrier with this system while 1.5g of lipid nanoparticles are administered intravenously into the body, greatly improving its penetration rate without borneol amine . These can prove that our design is feasible. In addition, these also prove that our design is superior to the existing brain drug delivery methods and can improve the efficiency and targeting of drug.

Figure 8. yield of BO-NHS-PEG-PLAG

Figure 9. the relation between concentration of lipid nanoparticles crossing the blood-brain barrier and time

References

[1] Markham, Kelly A et al. “High-efficiency transformation of Yarrowia lipolytica using electroporation.” FEMS yeast research vol. 18,7 (2018): 10.1093/femsyr/foy081. doi:10.1093/femsyr/foy081

[2] Ji, Qingchun et al. “Improving the homologous recombination efficiency of Yarrowia lipolyticaby grafting heterologous component from Saccharomyces cerevisiae.” Metabolic engineering communications vol. 11 e00152. 13 Nov. 2020, doi:10.1016/j.mec.2020.e00152

[3] Zhang, Nini et al. “Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.” Microbial cell factories vol. 20,1 224. 14 Dec. 2021, doi:10.1186/s12934-021-01715-y

[4] Wong, Lynn et al. “YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica.” Metabolic engineering communications vol. 5 68-77. 1 Oct. 2017, doi:10.1016/j.meteno.2017.09.001

[5] Joseph Sambrook, David W.Russell Molecular Cloning：A Laboratory Manual[M]