Part:BBa_K3053042
Beta lactamase mRNA targeting sequence
This part is the culmination of all that we have done. This will produce the antisense mRNA which is designed to target the beta lactamase gene in pBR322 which confers antibiotic resistance against the antibiotic ampicillin.
Usage and Biology
This part works when there is the beta lactamase gene in the organism it is transformed with. Under this condition, the bla (shortened beta lactamase) mRNA is targeted by the mRNA produced by our part. This forms a double stranded mRNA covering the RBS region of the bla which causes the bla mRNA to degrade.
As a result of this, no beta lactamase is produced removing the bacteria's resistance to ampicillin. In the absence of a bla gene the sense part of our construct BBa_K3053069 (https://parts.igem.org/Part:BBa_K3053069) forms a self complementary stem loop and ensures its further degradation.
Methods and Validation
The working of our part can be proven in a very simple manner. Since our construct targets the bla mRNA which confers ampicillin resistance to the bacteria, its activity is bound to ideally resensitize the bacteria to the antibiotic. However practically, we expect it to significantly reduced the number of resistant bacteria.
To prove our methods, we first transformed E.coli cells with pBR322 which contains the bla gene. The growth in this plate is plentiful (image below).
The next step is to transform the E.coli cells with pSB1C3 which contains our construct. The plasmid contains a resistance gene for chloramphenicol which will act as our selection marker. Similar to the first transformation, ample growth is present in this plate as well. An image is shown below:
The double transformants (containing both pBR322 and pSB1C3+construct) were produced by culturing the bacteria from the first plate and transforming them with the pSB1C3 containing our construct. They were then selected by growing them in a media containing both tetracycline and chloramphenicol and another containing ampicillin, tetracycline and chloramphenicol. The former will confirm double transformants while the latter confirms our part activity. As expected, lower growth is seen in both the plates as the efficiency of a double transformation is very low.
To obtain the pure colonies of the double transformed, a subculture was performed using the colonies from the Chloramphenicol+Tetracycline plate. An increased growth was observed this time. These colonies were then transferred to a plate containing ampicillin, chloramphenicol and tetracycline. This way we can confirm the working of our part. If the colonies grow, then our system does not work and the if they don't grow or show very low growth, they are a clear indication of our part working. And this is exactly what we observed during our experimentation. The A+C+T contained little to no growth.
For more information, please do not hesitate to check our Experimentspage and Resultspage.
Sequence and Features
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