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.

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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'