Proteins that Glow.

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Today we got back the results of our DNA sequencing. And it confirmed that we had indeed inserted our DNA fragment!  After two attempts, this is great news.  Not because we have now synthesised a protein.  But because it allows us to move forward with our experiment and attempt to do so.  First, however, to our positive result.  As with our earlier attempt to insert the fragment into the plasmid, once we had completed the insertion step, and thought it likely that we had successfully done so, we sent a sample of our plasmid with our putated fragment insert to a commercial company for sequencing.  And as with last results we obtained,  the sequence of our plasmid was visualised as a chromatogram and sent to us via email.

While the plasmid is sequenced by a commercial laboratory, the company does not confim or otherwise, whether or not we successfully inserted the DNA into the plasmid.  It just sequences the plasmid; and the reseacher(s) interprets the results.  As Cristiano knew the sequence of the DNA fragment we inserted, he was able to readily locate the order of the bases, and identify it within the full plasmid sequence that was returned to us.

Finally having the confirmation we need that we inserted the fragment, we can now proceed to remove it.  The fragment was only a dummy:  a spacer to prepare the plasmid for the DNA we actually want to insert.   I say fragment and plasmid but actually it is a case of fragments and plasmids: we prepared many more than one plasmid -in a single insertion process – with a dummy fragment and will insert a different DNA sequence into many more than one plasmid.  The DNA fragments which we will insert are known as NBP’s (Never Been Born Proteins). NBP’s are a randomly produced library of DNA sequences that code for amino-acid chains that haven’t been selected for by nature.  Potentially, these amino acid chains fold into protein structures by which they could become functional.  However, only a small sample of all possible protein sequences and are thought to have been sampled by nature. Using a 50 residue polypeptide as  a relatively short protein sequence that may be formed out of a combination of twenty known amino acids, there are ten to the power of 65 possible protein sequences -which is an astronomical number -which may occur.  Increase the length of the sequence and the number increases even more.  Due to the vastness of possible amino acid sequences, and the relatively small of amount of known extant proteins, it is hypothesised that nature could not have possibly sampled all possible amino acid sequences, in its selection of those that exist.

By randomly producing new DNA sequences, the Luisi Lab has developed new proteins from the vast possiblities of sequences that have not been selected for by nature.  However, while new amino acid sequencs can be produced it does not necessarily mean they will fold into functional proteins.  While the amino acid sequences are structurally expressed (in cells), there is no immediate way to know whether any or all amino acids sequences have actually folded into structured proteins. Therefore, a secondary means is currently neccesary to identify whether the folding process has taken place.

In the experiment we are currently doing, we are attempting to integrate the possible expression of the amino acid chain into a folded structure with the means by which it is identified.  Using GFP (green fluorescent protein)  -itself a functioning protein structure -inserted into the plasmid DNA as a folding reporter, in relation to which we positioned our NBP insert so that if forms part of the DNA sequence to be transcribed and translated into an amio acid chain, we hope to be able identify whether we have structured NBP’s: if the NBP’s have been inserted, as they are proteins which function when structured, they will glow.

Today we cultured the first cells with our NBP-eGFP inserts.  But will they glow?

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