tuberculosis potential cures through synthetic biology
TRANSCRIPT
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TUBERCULOSIS
POTENTIAL CURES THROUGH SYNTHETIC BIOLOGY
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• TB is caused by a mycobacterium (Mycrobacterium tuberculosis)
• Can be active or latent
• Latent TB forms granulomas in the lungs
TUBERCULOSIS FACTS
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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
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• 1/3 of the world population has latent tuberculosis
• Results in 1.3 million deaths per year
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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
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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.
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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
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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.
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PROCESS/EXPERIMENTATION• 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.
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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.
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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.
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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
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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:
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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
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Here is the general scheme for a phage that releases a silencing sRNA onto a cell containing genes for an antibiotic resistance.
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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