The battle against infectious diseases has always been painful and tough for
humans. One in every three global deaths can be attributed to an infectious
disease. According to the Global Burden of Disease study, almost 1 million people die from HIV/AIDS each year,
and more than 1 million sexually transmitted infections
(STIs) are contracted daily[1]. Apart from that, lower respiratory infections remained the world’s most
deadly infectious disease, ranked as the 4th leading cause of death [2]. Every year, it is estimated that influenza accounts for 400,000 respiratory fatalities worldwide, significantly hampering societal productivity [3]. The World Health Organization (WHO) also reports that up to one-quarter of the global population has latent
tuberculosis[4]. Since many infectious diseases are fast progressing and
highly contagious, nucleic acid testing (NAT), one of the most important diagnostic tools, becomes
increasingly essential. The prevalent technique used in China is qPCR, using fluorescent reporters and a PCR reaction to
amplify and detect specific genes [5]. However, the process is quite time-consuming and
energy-consuming due to the centralized testing of samples and repeated temperature changes for
amplification.
Recognizing the limitations of current NAT technology and the results of our survey (more than
65% of people hope the time for whole testing could be reduced to less than 1 hour), we have the motivation to innovate. After the research, we found that three naturally
existing phosphorothioate (PT)-DNA-dependent restriction enzymes may help us
with their binding and cleavage parts. However, the naturally extracted enzymes cannot be enriched
in the absence of histone tags.
Figure 1 Results of the questionnaire (dry team)
Figure 2 Top 20 death causes in the world 2023［2］
Phosphorothioate (PT)-DNA-dependent restriction enzymes are composed of
two domains: SBD and HNH. SBD could recognize and bind to sulfur modification on specific DNA fragments.
At the same time, HNH could cleave the DNA sites 8-15bp from sulfur modification when it’s activated as the ssDNA becomes dsDNA.
We selected the gene sequences of three related proteins called Sga,
Sva, and Asp.
To obtain the proteins, we used restriction enzymes Nde1 and Xho1 and
T4 ligase to construct three recombinant plasmids (Sga-pET28a, Sva-pET28a, Asp-pET28a). They were then transformed into DH5-alpha (a competent cell used
for cloning) and cultured in a petri dish.
An examination using restriction enzyme digestion was vital to verify
the construction condition of our genetic constructs. In our assay, the presence of both reporter genes
and vectors in each respective gel lane indicated successful digestion and confirmation of our
construct.. Given that the DH5-alpha competent cells are not suitable for
protein induction, we opted to transform our plasmids into BL21 cells, which are frequently used for
protein expression. [6], We then added IPTG, a molecular analog of allolactose, to three
separate LB broths. IPTG acts by removing a repressor protein from the lac operon, thereby inducing gene
expression.
After that, an ultrasonic cell disruptor was used
to lyse the bacteria, and we successfully got three reporter proteins.
To purify them, nickel affinity chromatography and
Q-column affinity chromatography were used to remove impurities, non-target proteins, and DNA attachment
step by step.
We then concentrated on three enzymes and did
SDS-PAGE, finding that both Asp and Sva failed in purification somehow. The exact concentration of Sga
was measured by nanodrop.
We designed two tests for protein function
analysis: EMSA and Nucleic acid cleavage, testing the binding and cutting abilities of Sga,
respectively. BL21 (The bacteria containing DNA naturally without sulfur modification) and B7A (The
bacteria containing DNA naturally with sulfur modification) were extracted and purified with a gravity
column, setting as two comparison groups. The results showed that Sga can perform these two functions on
phosphorothioate (PT)-DNA. It truly proved that the enzyme we designed has
the potential to be used in nucleic acid detection.
In the future, not only can this enzyme be used in
Point-of-care Testing, but it can also be used in environmental micro-organism detection and food safety
reservation. As both the operation time for the whole process (less than one hour) and energy used is
low, we believe we could achieve the goal of high efficiency of nucleic acid detection and reduce the
prevalence of infectious diseases worldwide in one day.
Figure 3. Work flow
[1]: World Health Organization: WHO. (2023). Sexually
transmitted infections (STIs). www.who.int. https://www.who.int/news-room/fact-sheets/detail/sexually-transmitted-infections-(stis)
[2]: deathmeters.info. (n.d.). Top 20 death causes in the World 2023 live — Deathmeters. https://deathmeters.info/
[3]: Dattani, S. (2023, May 18). Influenza. Our World in
Data. https://ourworldindata.org/influenza
[4]: Dattani, S. (2023b, September
5). Causes of death. Our World in
Data. https://ourworldindata.org/causes-of-death
[5]: mbl-Ebi.(n.d.).Real-time PCR | Functional genomics
II. https://www.ebi.ac.uk/training/online/courses/functional-genomics-ii-common-technologies-and-data-analysis-methods/real-time-pcr/
[6]: How does IPTG induction work? | GoldBio.
(n.d.). https://goldbio.com/articles/article/how-does-iptg-induction-work