Designed by: Kendall Kearns Group: iGEM12_UCSF (2012-10-02)
T7Prom+HisTag+YefM
This part contains YefM the antitoxin of the yefM-yoeB toxin-antitoxin system. It is behind a T7 Promoter with a , lac operator, ribosome binding site, and 6x his tag. The construct can be moved to any plasmid for expression.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 123
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Usage and Biology
Within the E.coli genome, there is the naturally occurring toxin-antitoxin system whose production is altered in response to various types of stress. In layman’s terms, a toxin-antitoxin system consists of two genes: one coding for the toxin, or “poison”, and one coding for the antitoxin, or “antidote”.
There are three different types of toxin/antitoxin systems, all with different products effectively committing apoptosis. A general overview of all types are listed below.
Type 1: Inhibition takes place when the antitoxin RNA binds to the complementary toxin mRNA. If there is not enough antitoxin RNA being transcribed, toxin proteins will be produced, inducing toxicity through cell membrane damage. Toxin RNA has a half life of ~20 minutes, while antitoxin RNA has a half life of ~30 seconds.
Type 2: both genes code for separate proteins, which bind to each other in a normal, unstressed state. While undergoing stressful conditions, the production of antitoxins will drastically decrease, allowing the toxin protein to act as a pseudo-RNAase, cleaving mRNA.
Type 3: The most recently discovered, inhibition of this toxin requires the interplay between a toxin protein and an antitoxin RNA gene. There is only one example of this system so far - the ToxIN system from the bacterial plant pathogen Erwinia carotovora.
YefM is a Type 2 antitoxin. For the purposes of the 2012 UCSF iGEM team, (tuning population ratios of symbiotic strains), Type 2 systems were determined to be ideal, since they have the greatest chance of longevity/sustainability as proteins, rather than RNA strands. In a Type 2 system (diagrammed above), the antitoxin gene is usually upstream of the toxin gene and its product is usually the more unstable of the two, degrading much more rapidly than the toxin. As this is the case, antitoxin proteins are produced in a much larger quantity in order to counteract the toxin. Antitoxin and toxin pairs are coded into proteins and bind to each other to prevent an accumulation of toxin. In stressful situations – when there is DNA damage, drastic change in temperature, or lack of nutrients – stress-induced proteases cleave antitoxins and leave the toxins to cleave the mRNA strands.
The 2012 UCSF iGEM Team used this part to determine if toxins and antitoxins could be used as a form of communication between two different strains of E. coli to tune population ratios.
We tried several different experiments to determine if the toxin an antitoxin pair could move between cells to cause an effect on growth. The final experiment we performed utilized protein induction at 30C which we believe helped maintain the stability of these small proteins.
A flask with 150ml LB+Spec was inoculated with either – Toxin strain, Antitoxin Strain, or BL21 (empty vector) control cells.
Each flask was started at an initial OD600nm of 0.05 and was grown for 2 hours at 37C before being induced with 0.5 ul/ml of 1M IPTG.
After the addition of IPTG, the flasks were transferred to 30C to promote stable protein production. These cultures were grown overnight, ~16 hours.
The next morning the cultures were centrifuged and the supernatant was filtered over a 0.2um filter to remove any cell debris, but retain any small proteins or molecules in the supernatant.
The filtered supernatant were then diluted (25ml supernatant + 75ml fresh LB) to provide a fresh supply of nutrients.
Then toxin, antitoxin, and control cells were inoculated into these filtered, diluted supernatant and induced with IPTG after 2 hours of growth at 37C.
The effect of supernatant potentially containing toxin or antitoxin proteins was observed in each strain.
The graphs shown below demonstrate growth of toxin (YoeB), antitoxin (YefM), and control cells in the media produced after step 5 above.
This first graph shows control cells that were grown in filtered supernatant from either Toxin, Antitoxin, or control cell growth. The inoculated control cells grow well in the supernatant from control and antitoxin cells but do not grow well in the presence of toxin supernatant. This indicates that the toxin supernatant actually contained toxins that had a growth effect on the control cells. This graph shows YefM, the antitoxin, being grown in filtered supernatant from control, toxin, and antitoxin cells. It is observed that the antitoxin grows well in each condition, except when inoculated into the supernatant where toxin cells had previously grown (green line).
This graph shows YoeB, the toxin, being grown in filtered supernatant from control, toxin, and antitoxin cells. It is observed that the YoeB toxin does not grow as well as any of the strains in the above graphs. However, there is a slight rescue of growth only in the case when it is exposed to the antitoxin supernatant (pink line).
