TUBERCULOSIS POTENTIAL CURES THROUGH SYNTHETIC BIOLOGY

TB is caused by a mycobacterium (Mycrobacterium tuberculosis) Can be active or latent Latent TB forms granulomas in the lungs TUBERCULOSIS FACTS

ANTIBIOTIC RESISTANCE Resistance to antibiotics has made treatment more difficult Diagnosis of resistance can take up to 3 months Curing tuberculosis takes between 6 and 20 months

1/3 of the world population has latent tuberculosis Results in 1.3 million deaths per year

DETECT One method uses a sensor to detect if bacterial DNA contains an antibiotic resistance gene A CRISPR/Cas system can bind to the target DNA and break it up This breakage induces the expression of a SOS sensor, which turns the cell blue Uses the DNA of a phage (phagemid) so that the system will spread throughout a population, turning it blue

This figure shows how a resistance gene causes the expression of Cas9 and gRNA, which guides the Cas9 to the specific resistance (in this figure, the kanamycin resistance) and it then generates a double strand break in the DNA of the phagemid. This causes the SOS response, resulting in a blue cell.

APPLICATION This technology could be most useful if it was incorporated into a tissue The system can test for both the presence of tuberculosis and an antibiotic resistance. The phagemid system would be incorporated into the tissue via an X-gal solution

INFILTRATE TB is an airborne disease, spreads through airways Macrophages in the lungs phagocytize the TB mycobacteria, but instead of dying, the latent bacteria can live on for years During this time, the bacteria can upset processes like phagocytosis for the macrophage The membrane of M. tuberculosis is waxy/thick with mycolic acid, which is hard for drugs to break through Synthetic biologists have attempted to create an E. coli strain that can penetrate the membrane and cytosol of the macrophage and deliver an enzyme to destroy the mycobacteria.

PROCESS/EXPERIMEN TATION Trehalose Dimycolate Hydrolase (TDMH) is an enzyme that degrades the mycolate layer and triggers lysis of the mycobacterial cell wall This was inserted into E. coli The modified E. coli were added to petri dishes with M. smegmatis and macrophages The goal was that the macrophages, if they ingested the M. smegmatis, would also ingest the E. coli so that the dangerous mycobacteria within the macrophage would be killed In one experiment, 99% of the bacteria were killed within 6 hours.

The macrophages were observed to have taken up the E. coli (red), the M. smegmatis (green), neither or both. Ideally, there wouldn’t be a macrophage that took up the mycobacteria that didn’t take up the E. coli, but the results are promising and scientists are still working to perfect the process.

APPLICATION Because TB primarily affects the lungs, the most efficient way to administer this technology would be through an inhaler. E. coli are small enough to pass through the alveoli and bronchioles, where macrophages with latent tuberculosis are present. There are about 600–800 macrophages per lung, which would require a dose between 10,000–100,000 E. coli.

SABOTAGE This method involves “sabotaging” cells with a synthetically-made virus Cells that have an antibiotic resistance are infected with a phage containing sRNA that silences the expression of the antibiotic resistance gene Antibiotic-resistant genes work by coding for antibiotic- resistant proteins If this process is stopped, the cells would be converted back to an antibiotic-sensitive state

PROCESS A device capable of silencing the gene resistant to antibiotics needed to be created Synthetic biologists used a “24bp sequence” to make this device Below is the structure of the chloramphenicol-resistant silencing device:

APPLICATION Can be spread through virus Overall burden of virus on cell is minimal When phages were released upon a population of kanamycin-resistant cells, 99.87% of the population was made sensitive again Issues: What if people avoided infection of the virus? Experiments showed that some cells developed a resistance to this sRNA after a time

Here is the general scheme for a phage that releases a silencing sRNA onto a cell containing genes for an antibiotic resistance.

CONCLUSION Can save millions of lives Are simpler and more likely to cure TB than current methods of treatment This is particularly important for people in lesser developed countries Greatest obstacle would be distribution and cost Some of these strategies need time to develop