PARTS

Grab hand

Construction of random library

The random peptide library we constructed is a kind of library with a large capacity, which can be used to screen short peptides and proteins. It should be noted that species do not limit the random peptide library. Through this platform, the researchers could screen the interacted small peptide of their protein of interest. So it is an extremely useful platform for screening protein-interacted peptides. This in-vivo screening method is also very feasible. The screened proteins can also be used again for yeast two-hybrid experiments and combined with bioinformatics analysis to find information on the critical binding sites to your protein. Our Random Peptide Library was finally constructed in pGADT7 GW after a Gateway BP-LR reaction (see also Design).

At the same time, to further test the capacity and characteristics of the random peptide library, we designed primers for amplifying the random peptide library sequences using the secondary plasmid library as templates. The resulting PCR products were subjected to illumine sequencing library construct. After sequencing on Illumina Novaseq 6000 platforms, the 60 bp reads were obtained and used for estimating the library capacity. As shown in the result section, our random peptide library is well constructed, and the library capacity is sufficient for screening. So we can announce the successful construction of our random peptide library and the addition of a new part.

Figure1. statistics of the sequencing data about our random peptide library. (A) Statistics of the peptide number of different lengths; (B) Saturation analyses according to randomly sampling from the sequencing data. The abscissa represents the proportion of random sampling including 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, and the ordinate represents the number of peptides detected corresponding to the percentage of sampled sequencing data.

Escape model

First, we fuse the polypeptide with the anchor protein, so that our "carrier" has the ability to "transport" the virus. When there is a virus passing through, E. coli will catch the virus by "grasping".

CheZ

The power of E. coli is provided by the rapid rotation of propeller like flagella. The flagellum motor can rotate clockwise or counterclockwise. Counterclockwise rotation produces directional/straight swimming of bacteria, while clockwise rotation makes cells adjust to a new direction and roll in place.[1]

CheY is a key regulator of flagellum movement response. When it is phosphorylated, it will cause the flagellum to rotate clockwise, and the phosphoprotein phosphatase CheZ will dephosphorylate CheY, causing the flagellum to rotate counterclockwise . Previous studies have shown that when CheZ is knocked out from the chemotaxis pathway, the tumbling of E. coli cells is dominant, and the overexpression of CheZ will inhibit the tumbling of E. coli cells. Let the coliform move faster in a straight line.[2]

Figure2:The E. coli chemotaxis system and its control of motility

EL222

EL222 is a blue light-regulated switch that allows positive regulation of target gene expression, consisting of a photosensitive transcription factor (TF), a transactivation domain by nuclear localization signal (NLS),[3]VP16 trans-activation domain, and a bacterial-derived protein EL222. When exposed to blue light (450nm), the EL222 monomer in this system is two-to-two dimerized, thus transcriptionally activates genes located downstream of artificial promoter C120 (BBa_K3570023).

Figure3:Blue light induction mechanism of EL222

Here, we place CheZ at the downstream of the blue light promoter PBind. When there is blue light, Escherichia coli will move rapidly and irregularly. When Escherichia coli enters the area without blue light, the movement speed of Escherichia coli will slow down, and even stop in the area without blue light in macro view. The final experimental result is that there are almost no bacteria (no pigment molecules) in the blue light area.

Pigment molecules are produced in areas without blue light.

However, since bacteria themselves produce CheZ, they will also move in the absence of blue light. This is why our final results show that the boundary between blue light and non blue light is blurry. So next we want to knock out the CheZ gene in E. coli. It can really stop in the area without blue light.

In the blue light area, because some E. coli will move disorderly, we also inserted a toxic protein (BBa K117000) that will cause E. coli suicide after EL222. Under the condition of blue light, Escherichia coli will autolyse under the condition of toxic protein. The virus fragments it carries will be attached by the next E. coli and transported to the blue light free area.At the same time, this suicide circuit also ensures that our "carriers" will not spill into the environment.

Figure4. The three above：Darkness nurtures The next three：bu lights 100μmol/m2/sCulture for 20 hours