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Result

Construction of random library

First,we Coding DNA sequences for random peptide library.

Name Sequence(5' to 3') Length
random petide sequences Cgtcggggacaactttgtacaaaaaagttggaacc(nnk)20taagacccaactttcttgtacaaagttgtgcggccgcc 134bp

Secondly, we design of primers for amplification the random sequence library. After that,the random sequence library was constructed on the primary vector pDONR207 by Gateway BP reaction. Then pDONR207-random petide was subjected to LR reaction with pGADT7-GW vector. Finally, we obtain the primary library plasmid (pDONR207-random peptide) and the secondary library plasmid (pGADT7 GW-random peptide).

Figure1. pDONR207-random peptide vector


Figure2. pGADT7 GW-random peptide vector


Figure3:Electrophoresis image of random peptide library fragments


Figure 4: Colony growth after bd-bait transformation into yeast ah109


Figure5: ah109 strain and y187 strain binding subs


Figure6: Growth of the transformed strain

Validation of a suicide system

In order to adapt our toxic proteins to the escape system, we made some modifications to the lytic protein.The best concentration is achieved by reducing the toxicity of the toxic protein.We have modified some amino acids so that the bacteria do not die too quickly when expressing the protein.In the end, we chose one option and transferred the gene into a Jilin University light-controlled parts. Finally,We grow this bacteria under the blue light condition.

Master and familiar with the safety of procedures and reagents to ensure that we strictly follow the general guidelines for safety management of Fujian Agriculture and Forestry Laboratory during the experiment (FIG. 1).

figure7:the suicide plasmid

The final results showed significant growth suppression within 10 to 20 hours after the insertion of the suicide gene, as we expected

figure8:The three above:Darkness nurtures The next three:bu lights 100μmol/m2/sCulture for 20 hours

Validation of two-component systems

To determine the detection limit and linear range of our cellular sensors. We established the relationship between the final fluorescence intensity and spike protein concentration by modeling.After obtaining our peptide library sequence, we transfered the sequence into the sensing unit of the PMRB (thanks to the strain provided by Tsinghua) , followed by the Spike protein after the optimized sequence adopted by the yeast doublet previously We did a series of gradient experiments, and the final results were similar to the modeling results.However, the detection limit was too high (that is, the sensitivity of the two-component system to the optimized spike protein was too low) . So we wanted to use peptides that could"Capture" Spike's properties to concentrate the virus.

Figure 9 Mathematical model of extracellular antigen concentration and fluorescence intensity

Validation of the escape system

To enrich our virus, we wanted to use the peptides to“Catch” the virus by binding specifically to the spike.But after catching the virus, we also need E. coli to be able to move in a given direction.For ease of use, we chose blue light as our trigger mechanism.

When irradiated with blue light, E. coli can move in a straight line at high speed because of the large amount of expression of this gene, but not in the region irradiated with blue light. E. coli rolls around because of the knock-out of the gene. After some time, E. coli will accumulate in areas without blue light. This is the“Light-repellent” we construct for E. coli. Take advantage of this property,E. coli then“Carries”the protein to areas where there is no blue light.

hACE2 SOEPCR

First, we simulated the structure of HACE2 based on the RBD with SARS-CoV-2 using UCSF Chimera and predicted the site of enhanced HACE2 For these sites, we designed corresponding primers to modify and synthesize the PCR productsWe recovered the pcr reaction solution and sequenced it to make sure that all the mutations were successful, then we recovered the fragments and constructed the vectors by infusion ligation. Finally we used the constructed vector to perform yeast two-hybrid, but unfortunately we found that the vector we designed did not show their ability to interact with each other in this way after performing yeast two-hybrid, so we communicated with the PI and wondered if the nls had not been added and thus no yeast two-hybrid did not show the results it should, so we modified the experiment again and gave our mutant sequence with the yeast nls fragment RGRGRGRGRGRGRGRGGYRGRARGFAPY*

Figure 10 The inter-site spacing between the key site before ace2 mutation and the RBD binding key atom of SARS-CoV-2


Figure 11 The spacing between the key atoms bound to the RBD of SARS-CoV-2 becomes smaller after mutation of the ace2 key site


Figure 12: Electrophoresis image of the sequences after point mutation

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