Ljubljana/methods
From 2007.igem.org
Parts Design
Since almost all of the parts used by our team had to be designed de novo, a huge portion of our time in the lab was spent on cloning. For each basic part, we designed primers containing standard BioBrick restriction sites. We had to introduce point mutations into some genes, because they contained one or more standard restriction sites inside the coding region. These mutations were introduced by PCR-driven overlap extension method (REFERENCA).
The genes, amplified by PCR, were therefore flanked by XbaI site upstream and SpeI, NotI and PstI downstream of the gene. After restriction, fragments were cloned into BBa_J52017, a vector already containing a eukaryotic terminator sequence downstream of the cloning region. This vector has previously been constructed by the Slovenian iGEM 2006 team.
In addition to basic parts, we also designed dozens of composite parts, mainly constructs expressing chimeric proteins. Composite parts were assembled by three-point ligations (REFERENCA), leaving 6 nucleotides long SpeI-XbaI mixed restriction site inbetween the two domains. Some of our composite parts contain as many as 6 basic parts. Such composite parts were constructed by repeating three point ligations.
Current Disease Treatment
One of the first AIDS therapeutics were nucleotide or nucleoside analogues (NRTI – nucleoside-analogue reverse transcriptase inhibitors) – pseudosubstrates, that are during reverse transcription integrated into viral DNA instead of nucleosides and thus block the transcription. These drugs were superseded by non-nucleoside inhibitors (NNRTI) that could inhibit reverse transcriptase by binding into the alosteric site of the enzyme. The third type of drugs is a family of HIV protease inhibitors. In most cases specific inhibitors are very similar to protease substrates - the only difference is that because they cannot be cut, they block the active site by remaining bound into it.
Weakness of all these therapeutics is that they are very sensitive to HIV mutations – HIV can easily mutate and thus become drug resistant. A combination of drugs is used to minimize HIV's potential to develop resistance to each individual drug in the mixture. Some of the drugs induce mutations that have negative effect on the virulence and such drugs can be used in spite of developed resistance.
We still do not have a cure for AIDS that would be insensitive to HIV mutations. Our project presents new ways of potential AIDS therapeutics. Our approaches can be considered independent of HIV mutations. We have set up a few of biological ambushes; if HIV manages to avoid them, we presume that it would not be able to infect the cell anyway.
Classes of Antiretroviral Drugs
Antiretroviral drugs are mostly inhibitors of different stages in HIV life cycle. They are targeted at different enzymes or events that are typical for HIV infection – entry of the virus into the cell, reverse transcription, polyprotein cleavage... and are divided into seven main classes (REFERENCA!):
Development
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