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?
![](https://static.igem.org/mediawiki/2015/1/19/UMDhokprotein.png)
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.
![](https://static.igem.org/mediawiki/2015/d/d0/RockEmUMD.png)
"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.