4th May, 2010

CSL Corporate Registered Head Office

45 Poplar Rd
Parkville
Victoria VIC 3052

Dear Mr Mc Namee, I am writing to you and the rest of the CSL board as I wish to
propose an effective method of producing the peptide needed for the vaccination
of the recent outbreak of disease. This proposal brings together methods
employed by the industry and also from research papers which have positively
tested new efficient ways of producing gene sequences of peptides. Having had
considerable contact with such procedures of production of chemicals via genetic
reproduction, I propose that by utilising PCR and ligation methods (Campbell et
al, (2010)), CSL can effectively produce enough of the peptide for use in
vaccinations across Australia.

Using analogue or conventional PCR methods (including usage of HindIII enzyme

and electrophoresis) (Blow, (2007)), if certain methods are followed will give
accurate and cost effective results within a short period of time (Ai-Sheng and
Quan-Hong et al (2004)). Utilising these methods would logically be best as the
amount of peptide produced via the PCR would be maximised, thus allowing for
the rapid distribution of antibiotics to the general public. This procedure in
essence requires the usage of PTDS (PCR based Two-step DNA Synthesis) ((Ai-
Sheng and Quan-Hong et al (2004)) which has been proven by scientists in the
Agro-biotechnology Research Centre of Shanghai Academy of Agricultural
sciences in 2004, to be much more effective at producing the desired gene or
gene fragment.

This method briefly entails the usage of digesting the sequence of DNA which
contains the code for the peptide, to allow for PCR to occur. After a number of
replications (the proposed method uses less oligonucleotide base pairs so as to
quickly produce the desired DNA sequence (Ai-Sheng and Quan-Hong et al
(2010))). By reducing the oligonucleotide length also decreases the margin for
error in the final replication of the peptide code, thus allowing for an effective
vaccination.

I recommend this method (detailed below) with digestion and ligation of the
replicated gene in order to incorporate into E. coli bacteria via transformation of
the plasmid gene (UQ School of biological science (2010). To identify the
peptides presence, incorporation of the gene sequence into the plasmid must be
between the lacZ gene, as this is used as an indicator in most cases (Campbell,
et al (2010)). Culturing these bacteria would provide enough peptide for a rapid
vaccination program and benefit both CSL and the general public. To harvest or
collect this peptide, using cracking solutions to lyse the E. coli bacteria and firstly
identify the presence of the peptide which should be apparent in the colonies
which do not appear blue (UQ school of biological science (2010)). These
colonies should have the peptide that CSL needs for the vaccination program and
can be produced effectively with minimal replication errors due to the proposed
method.

I strongly recommend that you accept my proposal as CSL will benefit from the
rapid production of the peptide and will be able to effectively vaccinate the
Australian public. I thank you for your consideration and time.

Regards,
Peter Snow

Step by step procedure:

1. Isolate the DNA sequence which codes for the peptide using PCR methods.
The method proposed is a two-step PCR based DNA synthesis method as
discovered by Ai-Sheng Xiong and Quan-Hong Yao of the Shanghai
Academy of Agricultural sciences. This method is similar to PCR, but turns
out the same number of replications but in a shorter span of time. The
method described by Ai-Sheng and Quan-Hong uses 60bp with 20bp
overlaps in order to break down the peptide and rejoin it through the PCR.

2. Once the desired amounts of replications are complete, E. coli bacteria

should be immersed in the solution which contains the peptide sequence,
in order to absorb the DNA through transformation (Campbell, et al
(2010)) and incorporate the gene for the peptide into the plasmid genome.

3. Allowing time for the ligation of the gene into the plasmid, preparation for
culturing the bacteria should be carried out in order to produce enough
peptide (UQ School of biological science (2010). Once this is done, a test
culture of bacteria should be grown, to check if the plasmid has been
incorporated (electrophoresis can also be used) (UQ School of biological
science (2010)). Once the bacteria with the gene sequence in the plasmid
is found, liquid broth should be used to quickly culture the bacteria with
the peptide (UQ School of biological science (2010).

4. After enough bacteria with the peptide are cultured, using cracking
solution and centrifuges to separate the matter and isolate the peptide will
allow efficient harvesting of the peptide (UQ School of biological science
(2010).

References:

UQ School of biological sciences BIOL 1020 practical manual

Ai-Sheng Xiong and Quan-Hong Yao

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA
synthesis method for long gene sequences