Questions Long Overdue & Go Around In The Head

Bet you all miss bio updates ahahahaha


Phagemid: pBluescript (Additional)

- a plasmid with a phage origin of replication. It can be used to generate ssDNA when co-infected (of the host) with a helper phage which will recognise the phage origin of replication to initiate the replication. The phagemid is replicated along with phage DNA and packed into capsid. Packaging" of pBluescript II phagemids containing inserts is efficient since the pBluescript II vector is significantly smaller


*amended*Why use phagemid?

(I took the below information from the patent for phagemid. It is the idea of phage display if you look it under wikipedia)

The special feature of phagemids which can be used for a presentation of particular products, viz. ligands, is the presence of a gene on the phagemids which consists of a fusion between the gene for the product to be presented and a bacteriophage coat protein gene. An example for such a bacteriophage coat protein gene is gpIII, the gene for the coat protein III (pIII) of phages M13 or fd. This results, on superinfection with an M13-like phage in the production of phagemid particles in the supernatant where the ligand is physically coupled via the bacteriophage coat protein to the gene which encodes the ligand.

By randomly mutagenizing the ligand gene at a specific site, the invention in question provides a library of phagemids containing tens of millions of ligand variants. The concept of the present invention is that some of these variant ligands will have specific binding affinities for a particular target molecule. The subset of strongly binding phagemids can be selected by, for example, binding on a surface coated with the target molecule, the poorly binding majority of phagemids being washed away. The specific strongly-binding population is then eluted by, for example, weak acid. This enriched subpopulation of phagemids can then be amplified by reinfection and cultivation in Escherichia coli. Successive rounds of such binding and amplification lead to the enrichment of a few variant ligands which exhibit strong specific binding characteristics for the target molecule.


(Flea Mel, I hope this takes care of your question on co-infection although the part on smaller virus escaped me)


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Insulin Production from Animal Sources: do we maintain the animals or just culture their relevant cells?

Cell culture because it is more easily maintained as no lab will bother with keeping livestock - we leave that to the professional. But animal cells are larger (need more nurients) and yield is low so compared to bacteria, maintenance is still high.

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RNase A/B/C/D/E/F/G/H?
We can find Rnases A to H, all with relatively different roles or found in different tissues.

The letter H is not attributed to anyone in particular :)

" RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strand complementary DNA (cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assays."http://en.wikipedia.org/wiki/RNase_H

- If I may bring in a cross-unit thingy, you might have realised that RNase H can be present to remove the RNA primers during DNA replication too although DNA polymerase is capable of the removal of RNA primer as well - keep to the latter for now pls.


Yes, I did not kid you. The letter H is important.

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Terminal Transferase: Can it keep on adding nucleotides?

TdT add to the 3-OH ends. The rate of addition of dNTPs and thus the length of the tail is a function of the ratio of 3´ DNA ends: dNTP concentration, and also which dNTP is used.
Industrial-wise, they have worked out the concentration for optimal results.