Micromon recombinant DNA techniques course
Paula Lima
Project No. 2009/768
Copyright Australian Seafood CRC, CSIRO and Flinders University 2010
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PROJECT NO 2009/768: Micromon recombinant DNA techniques course
PRINCIPAL INVESTIGATOR:
Paula Lima
ADDRESS:
CSIRO Marine and Atmospheric Research
C/-CSIRO Livestock Industries
306 Carmody Road
St Lucia QLD 4067
OBJECTIVES OF RESEARCH TRAVEL GRANT
To travel to Melbourne and participate of the Micromon recombinant DNA techniques
course at Monash University, in order to learn relevant molecular biology techniques
and skills essential for the progress of my PhD studies.
NON TECHNICAL SUMMARY
Monash recombinant DNA techniques course is widely recognised as the leading
course of its type in Australia, which consists of a series of tutorials, lectures and
hands on laboratory work. It is set out so that lectures and tutorials are given prior to
laboratory work, to give background and details of the techniques that will be used.
Another highlight of the course is the tutor to student ratio, which 1:4 for the tutorials
and laboratory workshops. As shown in the course schedule presented toward the
end of this report, it is very intensive with the majority being hands on. Thus, as my
PhD project relies heavily on advanced molecular skills, I found that would be
significantly profitable to undertake formal training in the theory and practical aspects
of various routine molecular techniques.
Some aspects of my PhD involving molecular techniques:

Development of real-time PCR tests to analyse expression of the delivered
vaccine constructs. This is essential in assuring that all 6 DNA antigens are
expressed following vaccination and will be used to make decisions regarding
efficacy or as a way of excluding non-productive antigens.

Development of real-time PCR assays for immune gene expression following
vaccination and vaccination/AGD challenge. Due to a lack of reagents, qRT-
PCR has become the standard way of measuring cellular responses following
vaccination. These assays are important in examining the underlying effects
of vaccination on the immune system.

Use of western blotting and ELISA to measure humoral antibody responses.
These standard assays are essential to examine the magnitude of the
immune response following vaccination and need to be undertaken to
determine if all the DNA antigens drive a functional response

Use of PCR, restriction digestion, ligation and transformation techniques in
order to build fusion gene constructs for in-vitro assays of gene expression,
protein translation and cellular trafficking.
OUTCOMES ACHIEVED TO DATE
Since I came back from Melbourne, I have been putting into practice, daily, what I
have learned during the course. Among the techniques that I have been currently
using are: RNA extraction, cDNA synthesis and PCR. In the future, other techniques
learned in the course will also be used, such as: restriction digestion, ligation and
transformation techniques, real time PCR, western blotting and ELISA.
BACKGROUND AND NEED
Amoebic gill disease (AGD) remains the number one health problem of the Atlantic
salmon Industry, costing c.a $20-25 million annually. Since 2002 CSIRO has been
working with industry on the development of a 6 antigen DNA vaccine for AGD.
Currently, the vaccine affords approximately 44% protection in an acute-to-morbidity
challenge system. However, despite the positive effects detected, is still unknown
whether all 6 delivered antigens are contributing to this increased protection. As a
result, my PhD project has as a main objective to examine the mechanisms of
protection afforded by the AGD DNA vaccine. By undertaking the Monash
recombinant DNA techniques course, I aimed to improve my knowledge in molecular
biology, which is crucial for the progress of my project. The skills learned during the
course are not only helping me during my candidature, but also will assist me in the
future when it comes to working in the industry, as researchers with both aquaculture
and molecular skills are rare.
RESULTS
The skills and knowledge gained in the course will result in research output in the
form of published papers and presentations at conferences. At the moment, I am
working on the samples related to the expression and translation of the 6 antigen
DNA vaccine. The result obtained will be presented at the Australasian Aquaculture
2010 Conference – Hobart, Tasmania. Another positive result achieved by my
attendance to the course, is that it attracts people from a wide range of backgrounds.
This way, I could establish contacts with these fellow researchers, who may assist
me not only in my PhD, but also in future opportunities.
PROJECT OUTCOMES (THAT INITIATED CHANGE IN INDUSTRY)
The nature of this work does not pose an immediate change for the Atlantic salmon
industry. However, as my PhD is part of a current Seafood CRC project – AGD
Vaccine Phase III (FRDC 2008/750), one of the 3 priority research projects funded
by the Tasmanian Salmonid Growers Association (TSGA), the knowledge gained
during the course will affected directly the progress of my project and, consequently,
the industry, as the commercialization of the 6 antigen AGD vaccine relies on the
results of the project.
ACKNOWLEDGEMENTS
I would like to acknowledge the Australian Seafood CRC, Flinders University and
CSIRO for funding the course and the trip’s expenses to Melbourne.
APPENDIX
Schedule of the course:
SUNDAY 15th
1.30
2.00
2.20
2.45
3.15
4.30
5.30
Arrival & collection of manual, etc
Welcome and information session
Meet your tutor for a laboratory introduction
Coffee/Tea Break
Lecture - Basic Microbiology
Lecture - Basic Cloning Requirements
Drinks & snacks
MONDAY 16th
9.00
9.30
Prelab
Restriction Enzyme Cleavage of DNA
10.30
10.45
12.30
1.15
3.30
3.45
6.00
Refreshments
Lecture - Gene cloning techniques I
Lunch
Restriction Enzyme Cleavage of DNA, ligation & examination of ligated DNA,
probe DNA
Refreshments
Transformation
Drinks & snacks
purification
of
TUESDAY 17th
8.45
10.00
10.15
10.45
12.45
1.30
2.15
3.30
3.45
5.30
Lecture - Hybridisation
Refreshments
Prelab
Colony hybridisation-preparation of probe, assessment of transformation results, colony
hybridisation-preparation of filters, Southern Blotting & preparation of target DNA
Lunch
Colony hybridisation of filters with probe DNA
Lecture - Gene cloning techniques II
Refreshments
Small scale plasmid preparation, Southern blotting and labelling of probe DNA
Drinks & snacks
WEDNESDAY 18th
8.45
9.45
10.00
10.30
11.15
12.45
1.30
2.45
4.15
4.30
6.00
Lecture - Polymerase Chain Reaction
Refreshments
Prelab
Miniprep of recombinant plasmids
Colony hybridisation-detection of signal, Southern Blotting-preparation of nylon membrane,
isolation of probe DNA and hybridisation
Lunch
Determination of the orientation of plasmid inserts, Cycle sequencing reaction
Lecture - DNA Sequencing
Refreshments
Lab continued
Drinks & snacks
THURSDAY 19th
9.00
9.30
10.30
10.45
1.15
2.00
2.15
3.30
4.00
4.15
6.00
6.30
Prelab
Preparation of cycle sequencing reaction, Southern blotting detection of probe DNA, RT-PCR
reaction, Induction of fusion protein production
Refreshments
Lab continued
Lunch
Lab
Lecture - Cloning Vectors
Polyacrylamide gel electrophoresis, PCR reaction -detection of strains
Refreshments
Western blotting
Drinks
Official dinner at the Wheelers Hill Hotel & special awards presentation
FRIDAY 20th
9.00
10.00
10.15
10.45
Lecture - Gene Expression Systems
Prelab
Analysis of PCR products, Western blotting protein immunoassay
Refreshments
11.00
1.00
2.00
3.30
Computer-aided analysis of Sequence data & demonstration of Real-time PCR, & Micromon
facilities including DNA sequencing/ Oligo Synthesis instrumentation
Lunch - presentation of certificates
Lab continued
Drinks & snacks