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When Alexander Fleming won the Nobel Prize in Physiology or Medicine in 1945 for his discovery of the first antibiotic penicillin, he acknowledged and warned the world that the misusage of antibiotics would bring a threat of antibiotic resistant microbes. Indeed, in the past decades, the occurrence of antibiotics resistance in microbes has appeared more frequently and it has become more difficult to treat these infections. Therefore, we aim to manipulate a bacterial immune system, CRISPR/Cas to combat antibiotic resistance.

The CRISPR/Cas system is the adaptive immune system presented in many bacteria, and it protects them from the invading genetic materials such as the viral DNA. When a foreign double stranded DNA (dsDNA) invade these specific bacteria, a portion of the DNA is captured by their system and incorporated into spacer sequence. Further invasion of the same dsDNA is recognized by the spacers incorporated earlier and destroyed by the CRISPR-associated protein, Cas3. Through engineering the spacer sequence, we can make the system target any dsDNA that is complementary to the spacer(s).

How are we going to make use of such intrinsic system to fight against the antibiotic resistant microbes? Bacillus subtilis could have their materials exchanged with both gram positive and negative bacteria through forming nanotubes. As B. subtilis lack of the CRISPR/Cas system, we need to engineer this immune system into B. subtilis. Upon the success of such implantation, the immune components could pass to the others’ cytoplasm through the nanotubes in between, and by manipulating the spacer sequence, the antibiotic resistance gene and any other DNA sequence of interest can be destroyed.

ABSTRACT

Although the sensory technology has been deeply explored and implemented in various means, most of the developed sensors are chemically-dependent promoters which regulate downstream gene expression. We exploited the use of halobacterial sensors, the sensory rhodopsins which are sensitive to a wide spectrum of readily available light source and build a series of sensing systems to control cellular movement and gene regulation. This system can be executed as a fundamental part for further applications, such as cell targeting and refining. Furthermore, to counter the safety issues caused by the leakage of bioengineered cells, this sensing method altogether with the CRISPR/Cas sytem can targart and achieve the cleavage of the transformed plasmid under the stimulation of natural light sources.

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