Team:GXU-China/Design

Engineering

It’s well known that tuberculosis is a serious chronic infectious disease. Its pathogen Mycobacterium tuberculosis is widely present in the human body. Whereas, since the outbreak of Covid-19, the medical and health resources of various countries have begun to be greatly tilted to fight against the major disease of the noble coronavirus. Therefore, this has also caused the weakening of the prevention and treatment of tuberculosis in various countries.

By consulting, we found the pathogen of tuberculosis is that the widespread existence of TB. Due to experimental safety requirements, we selected Mycobacterium smegmatis, which is similar to Mycobacterium tuberculosis, as the research object. Furthermore, we chose a specific phage selected from nature as the biological material and it can restrain the growth and reproduction Mycobacterium smegmatis.

So, we hope to construct a special Mycobacterium smegmatis phage through this experiment. Its capsid protein will be attached with the antigen protein of SARA-Cov-2 as a fusion protein. The injection of this particular phage will achieve two kinds of effects: Inhibiting the reproduction of 了，ycobacterium smegmatis in the body by cracking the host bacteria; using the SARS-Cov-2 antigen to stimulate and cause the body's immune effect to prevent the noble coronavirus. So as to achieve the idea of "one needle with two uses".

Experimental materials

1. Biological materials:

1.Mycobacterium smegmatis Mc2155
2.Ysogenic bacteria SDjn4 (Mc2155::SDjn4)
3.Bacteriophages SDjn4
4.Escherichia coli DH5ɑ
5.Three different types of SARS-CoV-2 virus antigen protein genes: Receptor binding domain (RBD) of Spike protein (S protein) gene, Envelope small membrane protein (E protein) gene, and Nucleocapsid phosphoprotein (N protein) gene
6. Three phage capsid genes fused with three different SARS-COV-2 virus antigen gene in C-terminal

EXPLANATION:

The above experimental materials 1, 2, and 3 are provided by the Laboratory of Microbial Synthetic Biology, School of Life Science and Technology, Guangxi University; The experimental materials No. 4 and No. 5 are all provided by BGI Gene Company with gene synthesis services

2. Experimental reagent：

1.Restriction endonuclease: Spe I (Takara Bio Inc., Japan) Hind III (Takara Bio Inc., Japan) Nhe I (Takara Bio Inc., Japan) Pac I (NEB Inc., USA)
2.DNA polymerase： TaKaRa TaqTM (Takara Bio Inc., Japan) PrimeSTAR ®GXL DNA Polymerase (Takara Bio Inc., Japan)
3.DNA ligase： T4 DNA Ligase (Takara Bio Inc., Japan)
4.Medium: Yeast Extract (Oxoid Ltd., UK) Tryptone (Oxoid Ltd., UK) DifcoTM Middlebrook 7H9 Broth (BD Co., USA) DifcoTM Middlebrook 7H10 Agar (BD Co., USA)
5.Reagent test kit: Gel Recovery Kit: Gel Extraction Kit D2500 (Omega Bio-Tek., USA)
6.Other reagents: BIOWEST® Regular Agarose G-10 (GENE COMPANY LTD., HK) Other reagents are purchased from Sinopharm Chemical Reagent Co., Ltd., China

3. Experiment equipment:

Thermal Cycler: Applied Biosystems Veriti™ Thermal Cycler (Applied Biosystems., USA)
Micro centrifuge: Centrifuge 5424/5425R (Eppendorf Co., Germany)
Nucleic acid electrophoresis: Mini-Sub Cell GT Horizontal Electrophoresis System and PowerPac Basic Power Supply (Bio-Rad Laboratories, Inc., USA)
Gel imaging system: ChemiDoc XRS+ System (Bio-Rad Laboratories, Inc., USA)
Electrotransformer: Eppendorf Eporator® (Eppendorf Co., Germany)

Experiment process

1. Plasmid construction：

a. Respectively, amplify the up and down stream of the SDjn4 phage integrase gene both about 1kb from the SDjn4 phage through PCR technique
b. Clone about 1kb downstream of integrase gene into Pac I and Spe I sites of pMind-lacZ plasmid, and clone about 1kb upstream gene of integrase gene into Hind III and Nhe I sites of pMind-lacZ plasmid
c. Clone the three fusion protein genes into the Spe I and Hind III sites of pMind-lacZ plasmids to complete the construction of the recombinant replacement plasmid

2. Fabrication of Escherichia coli Transformation Competent：

a. Pick a single colony of Escherichia coli DH5ɑ by streak plating and inoculate in 3ml LB liquid medium. Then culture oscillating on a shaker at 37℃ at 160rpm/min
b. Inoculate the DH5ɑ bacterial solution that has been cultured overnight for 1% ratio of volume in 100ml LB liquid medium, and culture with shaking at 37℃ for 2-3h（Till OD600=0.5-0.6）
c. Pre-cool the bacteria liquid in the mixture of ice and water for 5-10 minutes. Then transfer the pre-cooled bacteria liquid to a centrifugal tube and place it on ice for 10 minutes to cool the bacteria liquid fully
d. Transfer the cold bacterial liquid into a 4℃ centrifuge at 6000rpm/min for 5min. Discard the supernatant and collect the bacteria
e. Add 60ml cold 0.1mol/L CaCl2 solution to resuspend the bacteria, and then cool it in an ice bath for 45min. f. Transfer the bacterial liquid into a 4℃ centrifuge at a speed of 6000rpm/min for 5min to discard the supernatant and collect the bacteria
g. Add 1 to 2ml cold 15% glycerol solution (containing 0.1mol/L CaCl2 solution) to resuspend the bacteria, and store at -80℃ after loading respectively

3. Transformation of competent state of E.coli:

a. Add an appropriate amount of plasmid or ligated reaction product to 100μl competent cells, mix slightly, and take an ice bath for 30min
b. The receptive state was placed in a water bath at 42℃ for 90s incubation
c. After incubation, place the competent cells quickly in the mixture of ice and water for cooling, and then add into 800μ L LB liquid medium for shaking culture at 37℃ for 1h
d. Appropriate amount of recovered bacteria solution was coated on LB solid plate containing corresponding antibiotics, and the culture was carried out at 37℃ overnight to observe the growth of transformants

4. Plasmid extraction of E. coli:

a. Inoculate Escherichia coli strains containing the plasmid in 3ml LB liquid medium containing corresponding antibiotics for 12-16h
b. Take 1-2ml bacterial solution, centrifuge to collect the bacteria, and discard the culture medium;br> c. Add 200μ L alkali lysate I to the bacteria and suspend fully
d. Then add 200μ L alkali cracking solution II, reverse and mix repeatedly, stand at 4℃ for 5min, centrifuge at 12000rpm/min for 5min
e. Transfer the supernatant to a clean centrifugal tube, add anhydrous ethanol of 2 times volume of the supernatant, mix and precipitate at 4℃ for 5min, then centrifuge at 12000rpm/min for 5min, discard the supernatant
f. Add 500μ L 75% ethanol solution to the precipitation, discard the supernatant after centrifugation, add an appropriate amount of sterile deionized water to dissolve after drying at room temperature, and store the plasmid solution at -20℃

5. Fabrication of the induction state of Mycobacterium smegmatis:

a. Inoculate mycobacterium smegmatismus Mc2155 in 3ml 7H9 liquid medium (containing 0.05% Tween-80) and incubate it at 37℃ for about 2 days
b. Inoculate the saturated bacterial solution into 100ml 7H9 induction medium (7H9, 0.05% Tween-80) at a ratio of 1%, and incubate at 37℃ for 16h
c. Incubate the bacteria liquid on ice for 10-20min
d. At 6000rpm, centrifuged at 4℃ for 5min to collect thall
e. After the supernatant is discarded, add 25ml 10% sterile cold glycerol (containing 0.05% Tween-80) to clean the cells, and this operation should be repeated for 3 times to fully wash the thallus
f. Centrifuge and collect the bacteria, suspend the bacteria in 1-2ml 10% sterile cold glycerol (containing 0.05% Tween-80), then divide the bacteria solution into 100μ L volume, store at -80℃. Thaw it on ice before using

6. Construction of recombinant phage:

a. Import the three constructed recombinant plasmids (PMIND-CS, PMIND-CE and PMIND-CN) into SDjn4 lysogenic bacteria (Mc2155 ::SDjn4) by electroporation (2500V)
b. Add 1ml 7H9 liquid medium to SDjn4 lysogenic bacteria cells after electroporation, and resuscate the cells at 37℃ for 30min~2h
c. Add 300μ L of wild MYCObacterium smegmatic Mc2155 culture solution to the resuscated culture solution, add an appropriate amount of the mixed culture solution to 5ml melted LB semi-solid medium (containing 2mM CaCl2 and 0.6% agarose), mix well and pour onto the 7H10 solid medium plate quickly
d. After the medium is solidified, the culture it overnight at 37℃. After that, observe the growth of plaque

How did we test it?

We performed a spot-on-lawn assay using both growth medium and cell lysate of the bacteria carrying different bacteriocin constructs and observe the inhibition zone on the plate.

Proposal

Finally, we have a target scheme for future experiments. As we all know, CRISPR is an immune defense developed by bacteria and archaea over a long period of time.While CRISPR technique is a kind of genetic engineering technology in which Cas nuclease is guided by RNA to modify targeted genes. Next, we will introduce CRISPR-CAS technology into the experiment. Using CRISPR as a bactericidal element, we will polish our special bacteriophage mentioned above so that it could more precisely target mycobacterium, thereafter preventing COVID-19 while better treating TB. For example,carrying genes encoding key proteins of mycobacteria on the CRISPR element allows Cas protein to targetedly knock out the genes encoding key proteins of mycobacteria, thereby enhancing its bactericidal effect.The realization of this vision will give our experimental project far-reaching significance.

Team:GXU-China/Engineering