Difference between revisions of "Team:UMaryland/Description"

Revision as of 18:34, 18 September 2015

Safe and Inexpensive Approaches to Advance Synthetic Biology

Alternative methods of plasmid maintenance and PCR amplification accelerate the construction of new biodesigns, reduce cost, and avoid environmental hazards. Plasmids are typically maintained in cells by encoding enzymes that hydrolyze or otherwise detoxify antibiotics added to the medium. However, this process carries an inherent risk for spreading antibiotic resistance to native bacterial populations through lateral gene transfer. The Hok-Sok toxin-antitoxin system, a natural internal maintenance cassette relying on internal mRNA silencing, presents an alternative to common antibiotic-based methods since it does not rely on exogenous drugs. We are also developing an integrated, microcontrolled thermocycler using common household components. Using nichrome wire and a motorized fan for air circulation, the programmable prototype is an inexpensive, versatile thermocycler or plate incubator. Because the material and construction costs are a fraction of dedicated instruments, the newly developed unit will find broad application among nascent synthetic biologists in underfunded environments.

Hok/Sok

While present on a plasmid, the Hok-Sok system maintains plasmids through mRNA silencing. The Hok-Sok cassette actually contains three genes:

1. hok - host-killing
2. sok - suppressor of killing
3. mok - modulator of killing

Hok encodes a 52 amino acid toxin that is capable of permeating the cell membrane, leading to a loss of electrochemical potential and cell death. The hok toxin acts with "single-hit" kinetics, indicating that a single molecule of translated hok is sufficient for killing a cell. Hok is located on the forward strand of the Hok-Sok cassette and is freely transcribed under a weak promoter. How then, do cells not die whenever they possess the Hok gene?

Hok: Small protein - Big effect

In order to stay alive, cells need to be able to block translation of the hok transcript. Located on the same locus as hok is mok, which modulates whether or not hok is properly translated by the ribosome. Mok is located on a different reading frame than hok and the two genes are always co-transcribed as part of a single mRNA transcript.

Mok is then recognized by sok, a short ssRNA "antitoxin" transcribed under a strong constitutive promoter on the reverse strand of the Hok-Sok cassette. Sok is capable of forming a double-stranded RNA duplex near the 5'-end of the mok transcript. This duplex formation indirectly blocks the hok open reading frame, preventing the transcript from being read. The dsRNA duplex is then recognized by RNAse III, leading to enzymatic degradation of the silenced transcript.

"Hok-Em, Sok-Em Robots"

Below is a simplified diagram of how the Hok-Sok system maintains a plasmid:

How does this process lead to plasmid maintenance? Hok mRNA, due to a high degree of secondary structure, has a long half-life, measured at 20 minutes. Sok, on the other hand, has a half-life of only 30 seconds. The cell must thus retain the plasmid, and thus, maintain the coding region for Sok if it is to produce enough Sok to continue blocking Hok translation. If the plasmid is lost, then both the Hok and Sok coding regions will be lost; however, previously transcribed Hok mRNA will still be present. With previously transcribed Sok rapidly degrading, Hok mRNA will be translated, killing the daughter cell that did not maintain the plasmid. In nature, this is how the Hok-Sok system maintains antibiotic resistance in E. coli in the absence of antibiotic pressure.

Our wet lab project for 2015 was to demonstrate that the Hok-Sok system could maintain a plasmid over many generations as well as typical antibiotic maintenance systems. We hypothesized that the Hok-Sok system could thus serve as an ethical alternative to antibiotic pressure.

Learn more about the Hok/Sok design

Thermocycler

PCR is a common tool used in the laboratory environment as it is a vital component in DNA amplification and any cloning procedure. However, the majority of PCR machines used in the lab are bulky and expensive and are rarely seen outside of biology-based labs. These issues inspired the development of UMD PCR, a compact PCR machine that is as fast and proficient at amplifying DNA as lab based machines, but constructible for the fraction of the price of lab-grade PCR machines and a great tool for teaching-based applications.

Learn more about our PCR design and development

References

Certain images used under Creative Commons license.

@@ Line 213: / Line 213: @@
 <p align = "center"><img src = "https://static.igem.org/mediawiki/2015/1/19/UMDhokprotein.png"></p>
 <p style = "font-size:18px" align = center><b>Hok: Small protein - Big effect</b></p>
-<p style="font-size:20px">In order to stay alive, cells need to be able to block translation of the hok transcript. Located on the same locus as hok is mok, which modulates whether or not hok is properly translated. Mok is located on a different reading frame than hok and the two genes are always co-transcribed as part of a single mRNA transcript.</p>
+<p style="font-size:20px">In order to stay alive, cells need to be able to block translation of the hok transcript. Located on the same locus as hok is mok, which modulates whether or not hok is properly translated by the ribosome. Mok is located on a different reading frame than hok and the two genes are always co-transcribed as part of a single mRNA transcript.</p>
 <p style="font-size:20px">Mok is then recognized by sok, a short ssRNA "antitoxin" transcribed under a strong constitutive promoter on the reverse strand of the Hok-Sok cassette. Sok is capable of forming a double-stranded RNA duplex near the 5'-end of the mok transcript. This duplex formation indirectly blocks the hok open reading frame, preventing the transcript from being read. The dsRNA duplex is then recognized by RNAse III, leading to enzymatic degradation of the silenced transcript.</p>
 <p align = "center"><img src = "https://static.igem.org/mediawiki/2015/d/d0/RockEmUMD.png"></p>
@@ Line 223: / Line 223: @@
 </div>
-<p style="font-size:20px">How does this process lead to plasmid maintenance? Hok mRNA, due to a high degree of secondary structure, has a long half-life, measured at 20 minutes. Sok, on the other hand, has a half-life of only 30 seconds. The cell must thus retain the plasmid, and thus, the coding region for Sok if it is to produce enough Sok to continue blocking Hok translation. If the plasmid is lost, then both the Hok and Sok coding regions will be lost; however, previously transcribed Hok mRNA will still be present. With previously transcribed Sok rapidly degrading, Hok mRNA will be translated, killing the daughter cell that did not maintain the plasmid.
+<p style="font-size:20px">How does this process lead to plasmid maintenance? Hok mRNA, due to a high degree of secondary structure, has a long half-life, measured at 20 minutes. Sok, on the other hand, has a half-life of only 30 seconds. The cell must thus retain the plasmid, and thus, maintain the coding region for Sok if it is to produce enough Sok to continue blocking Hok translation. If the plasmid is lost, then both the Hok and Sok coding regions will be lost; however, previously transcribed Hok mRNA will still be present. With previously transcribed Sok rapidly degrading, Hok mRNA will be translated, killing the daughter cell that did not maintain the plasmid. In nature, this is how the Hok-Sok system maintains antibiotic resistance in <i>E. coli</i> in the absence of antibiotic pressure.</p>
-<p style = "font-size:20px">Our wet project for 2015 was to demonstrate that the Hok-Sok system could maintain a plasmid over many generations as well as typical antibiotic maintenance systems. We hypothesized that the Hok-Sok system could thus serve as a cheap, ethical alternative to antibiotic pressure.</p>
+<p style = "font-size:20px">Our wet lab project for 2015 was to demonstrate that the Hok-Sok system could maintain a plasmid over many generations as well as typical antibiotic maintenance systems. We hypothesized that the Hok-Sok system could thus serve as an ethical alternative to antibiotic pressure.</p>
 <div style="text-align:center;">

Difference between revisions of "Team:UMaryland/Description"

Revision as of 18:34, 18 September 2015

Hok/Sok

Hardware

IL Study

Side projects