Engineering Success
SOE
Design
We used a model to predict eight point mutant sequences with stronger interactions with the RBD structural domain of the new coronavirus by bioinformatics on the original sequence of ACE2.
Table 1.ACE2 point mutation predicted enhancement sites
| Number | Spike binding sites | ACE2 | Distance change before and after mutation | Binding energy | Predictions |
| 1 | SARS-CoV-2 | hACE2 | Original sequence | -14.38 | / |
| 2 | Q493 | l_21_V | 6.541->6.506 | -14.15 | Stronger |
| 3 | F486 | K_26_R | 5.132->3.8007 | -14.38 | Stronger |
| 4 | F486 | T_27_A | 9.886->9.387 | -14.39 | Stronger |
| 5 | L455 | K_31_R | 13.985->11.889 | -14.37 |
stronger |
| 6 | F486 | N_33_I | 8.529->8.309 | -14.37 | Stronger |
| 7 | F486 | H_34_R | 15.184->12.349 | -14.36 | Stronger |
| 8 | F486 | E_37_K | 15.175->10.415 | -14.35 | Stronger |
| 9 | F486 | T_92_I | 7.047->7.0247 | -14.38 | Stronger |
Construct
Then we had the company build the original Ace2 sequence we designed on the pGADT7 vector, and synthesize the primers we designed for point mutation, and then mutate the original ACE2 sequence.
Figure1. pGADT7-ace2
Testing
After getting the plasmid constructed by the company, we mutated the synthesized pGADT7-ace2 which was combined with primers, and finally sent the mutated fragment to sequencing, confirmed that the mutation was correct and then ligated into the enzymatically cleaved pGADT7 by infusion technology, and then performed yeast double hybridization. (Figure: sequencing results, ligated electrophoresis, yeast double hybridization)
Analysis and Learning
Finally, we found that the results of yeast double hybridization were not obvious, and we suspected that there was a problem in the vector sequence construction, we added the nls sequence of yeast system RGRGRGRGRGRGRGRGGYRGRARGFAPY* to the sequence of point mutation in the subsequent experiments to conduct another experiment
Random Peptide Library
Design
In order to find the peptide with strong interaction with spike, we constructed a platform that can generate 20 amino acids randomly. And a series of peptides are generated by an exhaustively exampled method. This approach can generate a database of large capacity. We perform yeast two-hybrid interaction screening for the peptides in this database, and we can get a series of peptides that can interact strongly with the target protein.We then compared the similarities on the ACE2 sequences of these peptides and other species by bioinformatics approach, wanting to find potential viral hosts.
By reading the papers, we found that the library capacity of the random peptide library would be relatively large, so we prepared to construct the random peptide library fragment to pGADT7GW vector by gateway method, so we first constructed the random peptide library fragment containing attb sequence.
Figure2. Random peptide library sequences
Figure3. pDONR207-random peptide vector and pGADT7 GW-random peptide vector
Construct
The random DNA sequences encoding the 20 peptides were synthesized by Shanghai Biological Co. (N represents A, T, G, and C, K represents G and T.) Twenty short peptides contain attb1 and attb2 sites in the upper and lower sides of the sequence, respectively To terminate the translation of the random 20 peptide, the TAA stop codon was designed after the random 20 short peptide. In order to amplify a random DNA fragment of 20 short peptides, we designed two additional primers for the random DNA fragment (synthesized by Shanghai Biological Co.) After obtaining the sequence, we performed PCR amplification immediately, and then recovered the sequence and transformed it into pDNOER 207 vector. By using gateway bp reaction, and finally transformed into pGADT7-GW vector by LR reaction
Figure4. random peptide Build process：bd-bait successfully transferred into yeast AH109 without self-activation
Testing
We first transformed the spike protein sequence into the AH109 strain and tested it for self-activation. After confirming the absence of self-activation, we co-transformed this AH109 strain with the Y187 strain containing the secondary library. Finally, Cotransformants are plated on SD/-His/-Leu/-Trp medium. After this screening, we obtained 36 positive strains, in order to analyze the protein sequence characteristics we obtained, we performed colony PCR and detected its sequence information by sequencing analysis.
Figure5. Random peptide library transformation results confirm DK2 as the strongest binding short peptide with covid-19
Analysis and Learning
These results allow us to conclude that the construction of a random peptide library will allow us to quickly screen for interacting proteins of relevance to them for rapid detection when preventing the arrival of the next outbreak
PmrCAB Two-component system
Design
The PmrCAB system was a system to sense Fe(III) in the outside world for Salmonella originally, where we envisioned replacing the iron-binding motif of PmrB (amino acids 34 - 64) with the sequence of a random peptide library we have obtained to be highlighted in green. The relationship between viral concentration and final fluorescence signal intensity was then established using different concentrations of the previously described spike protein.
Figure6. Design and construction of a lanthanide-responsive system based on the PmrA/PmrB two-component system
Construct
We have changed the specific sequence of AffiPmrB in the strain provided by Tsinghua, replacing the binding motif with the sequence of our random peptide library.
Testing
To establish the relationship between final virus concentration and fluorescence intensity, characterization experiments were performed as follows:
When the bacteria were grown to the appropriate stage, IPTG was added to induce the expression of PmrB and PmrA. A certain gradient of spiked protein (after optimization), corresponding to three types of coronaviruses, was taken to stimulate the sensor PmrB to turn on a series of responses afterwards. Each concentration corresponds to one fluorescence concentration.
We also used a modeling approach to establish a theoretical relationship between virus concentration and fluorescence intensity.
Figure7:Relationship between spike protein concentration and fluorescence intensity(left：spike induction results under fluorescence microscope. The engineering bacteria was induced with 1.0 μg/mL~3μg/ml spike(after codon optimization). Photos were taken under fluorescence microscope.right：the theoretical relationship between spike protein concentration and fluorescence intensity was made by mathematical modeling method based on the principle of two-component system)
Analysis and Learning
We compare the experimental results with the modeling results. It was found that the general trends were in accordance with each other. However, the specific amounts differed considerably, which may be due to the wrong prediction of our peptide library and spike binding capacity parameters, and our modeling did not take into account factors such as bacteriophage concentration.
However, we can roughly conclude that the detection limit of our cell sensor is about 1 μg/ml, which is not sufficient for virus detection in wastewater. So we thought we needed to concentrate the virus before detection.
Suicide part
Design
To prevent our bacteria from overflowing into the environment. We designed the circuit for light-coupled suicide, which needs to be used together with our subsequent circuit, which requires us to precisely control the experimental conditions to determine the time of bacterial death after the light-controlled conditions are turned on.
To this end, we figured out the mechanism of bacteriocin cleavage in E. coli by modeling and wanted to obtain less toxic proteins by individual amino acid transformations. However, after comparing several options, we chose the original one and a blue light intensity of 150 HZ.
Construct
We changed the mRFP part of the previous plasmid to BBa K117000 element and cultured it.
Testing
We incubated the bacteria with blue light irradiation after coating the plates. And another batch of bacteria was cultured under the same conditions by avoiding light, and finally these two batches of bacteria were cultured under the same conditions.
Figure8.The three above：Darkness nurtures The next three：bu lights 100μmol/m2/sCulture for 20 hours
Analysis and Learning
We can find that the growth of bacteria is significantly inhibited under blue light, while bacteria not irradiated with blue light have no effect. This shows the success of our light-coupled suicide circuit.
light-coupled suicide circuit. We also found that in the already grown bacteria did not die by lysis in a large area under the condition of blue light irradiation, but we found that blue light did not significantly inhibit the growth of E. coli in our project last year. There may be a mechanism that we are not aware of.
the Escape Part
Design
To be able to concentrate virus in wastewater, we constructed an engineered bacterium capable of "transporting" the virus. We used light control elements to regulate the expression of a flagellar protein kinase. This kinase dephosphorylates CheY, causing the flagellum to rotate counterclockwise. This change causes E. coli to move forward at a faster rate. When CheZ is not expressed in the exogenous gene, the flagellin in E. coli will rotate clockwise, causing the E. coli to "roll" in the same place.We want to build the "light avoidance" of E. coli, when there is blue light exposure, E. coli will move at high speed to find a "way out", otherwise it will make too much virulence protein expression that would cause E. coli lysis and death if it is exposed to blue light for a long time. Thus, E. coli can only "carry" the adherent virus "escape" to the area without blue light.
Figure9.The mechanism by which E. coli "transport" viruses
Testing
We implanted the "escape system" in the above constructed E. coli with the suicide gene and implanted pigment proteins to indicate the location of the E. coli. Then we constructed a part of the blue light-free area with tin foil. Then we incubated with blue light.
Analysis and Learning
The final biological image has blurred border between the region without blue light and the region with blue light, probably due to the fact that we did not knock out CheZ in E. coli, which made some of the E. coli in the region without blue light escape back to the region with blue light.
