Results

Introduction

This page will give a detailed record about the experiment results, future plans & project achievements.

Experiment Results

Yeast

Summary of Lab Design

In order to decorate the yeast surface with nanobodies against the pathogen, we designed the vector which contains the nanobody gene behind a membrane-sealed protein. The overall experiment design could be divided into 3 phases. In phase I, the expected vector that could potentially express our designed module would be constructed and stored. In phase II, the expression vector would be transformed into yeast and detected for its expression abundance and location. In phase III, the binding affinity and inhibitory efficiency of the pathogen would be tested. Due to the limitation of materials and time under the background of the local pandemic situation, the experiments only accomplished Phases I & II.

Genes of nanobody and pathogen were harvested

The DNA sequences of nanobody 20ipaD and antigen IpaD were obtained from Sangon in the form of pUC19 plasmid insertions, which were stored inside E. coli. The storage bacteria were amplified, and plasmids containing sequences of interest were extracted with Plasmid mini-prep kit from TIangen. Those plasmids were digested by XhoI and ApaI, and the sequences of interest were recycled by agarose gel recycle kit from Beyotime (Fig1).

Recombinant plasmids were constructed

The purified IpaD and 20ipaD DNA segments mentioned in the previous section, along with the pYD1 vector digested by XhoI & ApaI, were ligated, and transformed into DH5α cells that were selected by ampicillin. Colony direct PCR was applied to inspect the plasmid condition of the colonies formed. pYD1-forward and pyd1-reverse primers were used to show the size of Aga2-Protein, thus verifying the sequence between the primer pairs (Table 1). The size of pYD1-IpaD plasmid primer box should be about 1500bp, while the size of pYD1-20ipaD should be about 1000bp. As shown in Fig2 a, samples "2-3", "2-5" meet the requirement for pYD1-IpaD, so the bacteria in those colonies contain the recombinant plasmid pYD1-IpaD. Additionally, all samples in lanes 1~10 met the requirement for pYD1-IpaD, which indicates the bacteria in those colonies contain pYD1-20ipaD recombinant plasmids (Fig2 b). Those colonies were amplified, and the plasmids were extracted. Sequencing data further supported the successful construction of the recombinant plasmids (Fig3).

Recombinant plasmids were transformed into yeast EBY100

We performed yeast transformation using constructed pYD1-IpaD and pYD1-20ipaD plasmids. Yeast competent cells were prepared by Frozen-EZ Yeast transformation II kit (Zymo Research, T2001). The yeasts were successfully transformed by the chemical method using carrier DNA and PEG/LiAc. The plasmids and competent cells were thawed on ice. 10μL of Carrier DNA and 2μg of plasmid was added into 100μL of competent cells. Then 500μL of PEF/LiAc solution was added. The tube was inverted 6-8 times and incubated at 30°C with an inversion at 10-minute interval. Then, 20μL of DMSO was added to increase the transformation efficiency. Then, the tube was incubated under 42°C with an inversion of 5-minute interval. The cells were centrifuged at 12000rpm for 5 seconds and incubated with YPD medium for 1 hour at 30°C. After incubation, the cells were pelleted and resuspended by 100μL of 0.9% NaCl, 50μL of which was spread onto SDCAA medium, and another 50μL of cells onto YPD medium. The plates were incubated at 30°C for 2 days.

The result of the plates was positive (Fig4). SDCAA plates, which are the selective plates that lack tryptophan, have demonstrated fewer colony growth compared to YPD plates, indicating successful transformation. Single colonies were taken and incubated at 30°C in SDCAA liquid medium for amplification.

Nanobody and antigen were expressed and displayed on yeast surface

The yeasts that were transformed with pYD1-IpaD, pYD1-20ipaD, and pYD1 were amplified in SDCAA liquid medium for 36h. The cells were harvested by centrifuge at 5000xg for 5 minutes and diluted in SGCAA liquid containing the induction agent galactose to obtain an OD600=0.5. Four milliliters of each diluted cells were transferred into new culture tubes and induced at 21°C for 24h. An additional prep of EBY100 cells without plasmids was also diluted to OD600=0.5 and induced in SGCAA for 24h.

The induced cells were harvested and washed with PBS. The yeasts containing pYD1-IpaD were inoculated with rabbit anti-HA tag antibodies, while the yeasts containing pYD1-20ipaD, pYD1, or no plasmids were inoculated with rabbit anti-FLAG tag antibodies and BSA. After being washed with PBS, the cells were inoculated with FITC-conjugated goat anti-rabbit immunoglobin antibodies and BSA without light. The cells were washed twice with PBS before being imaged under UV light. As Fig5 shows, the yeasts containing pYD1-IpaD and pYD1-20ipaD emitted green fluorescence, whereas the yeasts containing empty plasmids, or no plasmids did not. This fluorescence pattern proved the yeasts indeed expressed and displayed the nanobody and the antigen on their surfaces, which announced the success of engineering.

Summary

Based on the gel results (Figure 1 and Figure 2), we could approximately know the success of constructing recombinant plasmids (pyd1-IpaD and pyd1-20ipaD). The lengths of 20ipaD gene and IpaD gene are around 1000 bps and 1500 bps. From Figure 3, we verify the sequence of IpaD gene and 20ipaD gene that we inserted into pyd1 empty plasmids. From Figure 4, we used EBY100, auxotroph yeasts to ensure the recombinant plasmids were successfully transformed into yeast cells because only cells with recombinant plasmids can survive on the Minimal Dextrose plates. Based on Figure 5, both IpaD antigens and 20ipaD antibodies were expressed on the surface of yeast cells because the preliminary antibody with FITC conjugated will connect to the tag expressed with IpaD antigens or 20ipaD antibodies on the yeast surface. FITC will be excited by the 488-nm light (blue light) and will release green lights (525/40 BP).

Lactococcus lactis

Escherichia coli

Summary of Experiment Design

We plan to display the nanobody JPS-G3 agianst the Shigella antigen IpaD on the surface of E.coli Nissle 1917 via the curli fiber protein csgA¹. We expect the engineered E.coli Nissle 1917 to be capable of capturing the pathogen Shigella flexneri via antigen-antibody specific binding.

Plasmid Extraction

pUC19-JPS-G3-3xHA and pUC19-IpaD-3xHA were extracted using the TIANGEN plasmid extraction kit.

Target Sequence Amplification

Using following primers (Table 1), the JPS-G3-3xHA and IpaD-3xHA sequences were successfully amplified, and the results were verified by gel electrophoresis (Figure 1). PCR products were purified for further manipulation.

Table 1 | PCR Primers Used

Figure 1 | Gel Electrophoresis Result for Target Sequence Amplification

Molecular Cloning for Cytoplasmic Protein Expression

In order to construct the recombinant plasmids pET30a-IpaD-3xHA and pET30a-JPS-G3-3xFLAG, the PCR products obtained from the last step were digested by endonucleases BamHI and XhoI to create sticky ends. Meanwhile, the pET30a plasmids were transformed into E.coli DH5a competent cells for propagation. After transformation and overnight culture, the extracted plasmids were also digested with endonucleases BamHI and XhoI to create sticky ends. Afterwards, the digested plasmids and target sequences were ligated and transformed into E.coli DH5a competent cells (Figure 2).

Figure 2 | Plates for pET30a-IpaD-3xHA (i-1 & i-2) and pET30a-JPS-G3-3xFLAG (J-1 & J-2).

Single colonies were picked from the plates and the inserted sequence were verified by colony PCR (Figure 3) and Sanger sequencing.

Figure 3 | Gel Electrophoresis Result for Colony PCR

Overlap Extension PCR

In order to construct the recombinant DNA sequences csgA-JPS-G3-3xFLAG and csgA-IpaD-3xHA, the following overlap extension PCR primers are used (Table 2).

Table 2 | Overlap Extension PCR Primers

However, due to time limitation, this part has not been successfully implemented.

Project Achievements & Analysis Summary

Successes

• Designed nano-antibody targeting the IpaD, a Shigella-exclusive antigen. Calculated the binding kinetics of the nano-antibody with the Shigella antigen in silico to verify the antibody design.
• Developed engineered bacterium (DH5α) and yeast (EBY100) that express the pathogenic antigen IpaD and targeted antibody on the cell surface, respectively.

Future plans

• Simulate the microenvironment of human GI system, including proper pH, temperature and oxigen level to determine the growth curve and induction efficiency, and the influence of the engineered yeast.
• The Ampicillin resistance gene has potential risk of horizontal gene transfer, which could cause potential harm to the original GI tract flora. Our future experiments plans to knock-in the "promoter-Aga2-20ipaD" cassette into the yeast genome using CRIPSR-Cas9.
• To test the antimicrobial effects on Shigella Flexneri, we are going to conduct hemolysis experiments.
• To test the safety and therapeutic efficacy of our product, we are going to conduct in vivo experiments in rodent model.

Lessons Learned

• Always use culture media and plates that are freshly prepared for the best results.
• Monitor the growth of the engineered micro-organisms, note all the changes, and analyze the reason why there is any inconsistency with the expectation.

Reference