1. Cloning'of'novel'luciferase'gene'from'deep3sea''
planktonic'worm'Tomopteris*helgolandica*as'a''
way'to'improve'visualization'of*in*vivo'protein''
dynamics'
'
The'study'of'in'vivo'protein'dynamics'is'growing'rapidly'due'to'
technological'advancement.'However,'current'methodologies'
have'many'limitations'and'there'is'a'strong'need'for'
improvements.'Bioluminescence'microscopy'is'a'promising'
technology'in'that'it'does'not'need'excitation'by'light'and'seeing'
photon'emissions'are'a'result'of'a'chemical'reaction,'results'are'
very'speciAic'and'quantiAiable.'However,'a'major'setback'in'
bioluminescence'microscopy'is'the'strength'of'the'signal'emitted.'
Whilst'genetically'enhanced'luciferases'have'improved'the'status'
quo,'variability'of'their'half3live,'and'variability'in'their'enzyme'
activity'remain'obstacles1.'Fundamentally,'there'is'a'need'for'
new'luciferases'that'can'overcome'the'limitations'presented'by'
existing'luciferases.'
The'successful'cloning'of'a'novel'luciferase'gene'will'ultimately'improve'on'
current'methodologies'available'for'visualizing'in'vivo'protein'dynamics.'Not'
only'will'this'add'to'the'colour'palette'of'available'luciferases,'it'will'also'
introduce'a'novel'emission'spectrum'that'may'enhance'multi3probe'analysis'
in'bioluminescent'microscopy.'Ultimately,'it'may'be'used'as'a'tool'to'enable'
scientists'to'identify'any'pathological'deviations'associated'with'in'vivo'
protein'dynamics,'resulting'in'the'potential'development'of'applicable'
therapeutic'agents.
'
'''We'aim'to'identify'and'clone'the'luciferase'gene'from'
Tomopteris'helgolandica,'determine'its'physical3chemical'
parameters'through'qualitative'and'quantitative'analysis'and'
compare'the'emission'signal'with'other'commercially'available'
luciferases.'
''
'
'
'''We'hypothesize'that'if'the'luciferase'responsible'for'generating'
bioluminescence'in'the'organism'T.'helgolandica'is'cloned,'it'will'
improve'on'current'methodologies'available'for'visualizing'in'
vivo'protein'dynamics.'
'
'
'!
3'SufAicient'funding'from'a'corporate'or'government'body'
3'Zooplankton'sample'from'eastern'tropical'PaciAic'Ocean'water'columns.'Two'
cruise'ships'are'expected'to'acquire'approximately'1000'samples5'
3'Biosafety'level'2'Laboratory'
3'Appropriate'equipment'to'perform'transformation'experimentation,'light'and'
confocal'microscopy'and'molecular'spectroscopy'
RISK'ASSESSMENT'
'
1.'Bachelor'of'Science'(Biotechnology),'School'of'Applied'Sciences '2.''Bachelor'of'Science'(Biotechnology),'School'of'Applied'
Sciences''3.'Bachelor'of'Science'(Biological'Science),'School'of'Applied'Sciences'4.'Bachelor'of'Science'(Biotechnology),'School'of'
Applied'Sciences'
Isolate Tomopteris
helgolandica
Samples may be deposited to
the plankton sorting and
identification centre3 for
isolation.
Obtain zooplankton
samples
Samples will be sourced from
water columns within the
south east pacific ocean.!
Comparison of light
emission
Value of the bioluminescent
product will be assessed by
comparing light emission with
current commercially available
luciferases.
Analysis of the toxicity
of the luciferin in foreign
cells
The luciferin will be isolated
and induced into E.coli to
ensure no harmful effects may
result.
Analysis of light
emission
Bioluminescence microscopy4
will be performed on the
transgenic product to
determine light emission
within living cells.
Isolation of the gene of
interest and
transformation
The coinciding gene of the
luciferase will be transformed
into a foreign organism using
a compatible plasmid vector or
expression vector.
Risks' Overall Risk' Risk Description' Risk Management'
Financial! Medium! Exceeding budget! Attempt to minimize the
budget as much as
realistically possible to
ensure funding is
adequate!
Legal! Low! Possible issues with
sourcing the organism.
Copy right law infringement!
Ensure all laws are
upheld. Seek legal advice !
Health and Safety! Medium! Injury in laboratory due to
unknown hazards or staff
unawareness!
Ensure all staff are aware
of safety regulations and
have had prior experience
In the field if they intend to
handle harmful products!
Environmental! Low! Damaging environment the
organism is sourced from!
Ensure this level of risk is
maintained. Estimate
possible damages,
implement new strategies
to prevent damage!
Quality! Low! Loss of key staff, new staff
un aware how to maintain
organism !
Have introductory class on
working with and
maintaining organism!
Consumer &
Reputation!
Low! Damaging social reputation! Ensure professional
standards are maintained!
Figure 2 – Colour palette of known luciferases1
1: Bauer, C.R. (2013). Bioluminescence Microscopy: New Avenues in Live Cell Imaging.
G.I.T. Imaging & Microscopy; 4: 32–34.
2: Wilson, T.V. (2011). How Bioluminescence Works. Available at:http://
animals.howstuffworks.com/animal-facts/bioluminescence3.htm
3: National Marine Fisheries Research Institute. (2013). Plankton Sorting and Identification
Center. Available at:http://www.sfi.gdynia.pl/?page_id=62.
4: Kammerloher, W, (2008). Bioluminescence microscopy for cellular level circadian analysis
in the suprachiasmatic nucleus. Nature Methods; 5.
5: Fernández-Álamo, M. (2000). Tomopterids (Annelida: Polychaeta) from the eastern
tropical Pacific Ocean. Bulletin of Marine Science; 67: 45-53.
'
2
OfM
REFERENCES'
EXPECTED'RESULTS'
ACKNOWLEDGEMENTS'
We!would!like!to!thank!our!Group!Mentor!Ian!Macreadie!for!his!great!wisdom,!
knowledge!and!encouragement,!and!the!course!coordinator!Susanne!Teppe,!
for!delivering!a!practical!and!constructive!course!that!has!ultimately!
empowered!us!with!enough!knowledge!and!con>idence!to!take!on!the!real!
world.'
INTRODUCTION'
AIM'
HYPOTHESIS'
Anita'Markovska1,'Sebastian'Bass2,'Alex'Baldassarri3,'Jean'Mbeng4'
PROPOSED'METHODOLOGY'
REQUIRED'RESOURCES'