RESULTS AND DISCUSSION

Nonbioluminescent soft corals exhibit bright coloration

Diversity in the coloration of marine Anthozoan species are attributed

to their expression of GFPs. Nonbioluminescent soft coral species have been

found to be of no exception and previous studies have already isolated GFP-

like homologues from such species.

Tentacles of three soft corals collected from Cartimar, Pasay were

observed to be brightly colored (Figure 6), particularly at their tips. This may

imply high potential of presence and localization of GFP-like gene expression.

Fluorescence imaging confirms presence

Figure 6. Nonbioluminescent soft coralofsamples
fluorescent
withparts under
potential GFP-
blue light (λEx = 450- 490nm)
like genes. (a) SC a, (b) SC C, and (c) SC D show brightly colored parts
implying high potential as sources of GFP.
Initial screening of samples was done through fluorescent imaging.

Whole mount fluorescence images of the samples were with filters at

excitation range 520-560nm (green) and at 455-490 nm (blue).The three soft

coral samples exhibited bright green fluorescence upon excitation at 455-490

nm (blue light). It was also noted that at this particular range of excitation,

fluorescence in SC A, SC C, and SC D was observed to be localized at the

tips of their tentacles - supporting the presumption that fluorescent protein

expression is responsible for the distinct, bright, coloration found at the tips of

the soft coral samples (Table 2). Imaging using the stereomicroscope (Oro,

19
2011) under excitation with blue light confirmed intense fluorescence at the

tentacle tips (Figure 7).

Live view

Figure 7. Fluorescence imaging reveals soft coral fluorescent parts.

Whole mount fluorescence images of SC A, SC C, and SC D were imaged
with filters at excitation range 520-560nm (green) and at 455-490 nm (blue).
Green fluorescence was observed for the three soft coral samples at
localized parts upon excitation with blue light. Fluorescent parts were also
imaged under a stereomicroscope upon excitation with blue light. (No
stereomicroscope image for SC A)

Table 2. Nonbioluminescent parts with bright coloration of soft coral

samples exhibit green fluorescence

Initial
GFP-like
identification Colored Fluorescent
Sample protein
based on parts color
present?
morphology

20
 tentacles:
SC A Goniopora. yellow green green +
at tips
tentacles:
SC C Catalaphyllia green +
green
tentacles:
SC D Ricordia bright yellow- green +
green tips

Samples stored in RNAlater® yield total RNA ready for cDNA library
construction

Presence of 28s and 18s rRNA bands from total RNA extracts from SC

A, SC C, and SC D were observed (Figure 8). This indicates successful total

RNA extraction from the three soft coral samples for subsequent cDNA library

construction.

-28s
-18s

Figure 8. Total RNA extracts from dissected parts

of soft corals samples stored in RNAlater® .Visible
distinct bands for the 28s and 18s rRNA (pointed) of
the three corals can be observed.

Successful amplification of partial 18s rRNA fragments of samples

suggests construction of intact cDNA libraries

After preparation of the 3’ cDNA libraries of the three newly collected

soft corals: SC A, SC C, SC D, and also of another sample SC1 previously

21
indetified as Sinularia sp., the 18s rRNA fragmentsof the samples were

amplified to check the integrity of the libraries constructed. Figure 9 shows

successful amplification of their 18s rRNA fragments yielding the expected

band of approximately 800 bp in size. Sequencing is still needed though to

confirm identity of the fragments.

Figure 9. Amplification of partial 18s rRNA gene fragments of

soft coral samples SC A, SC B, SC C, and Sinularia sp. using
F1 and R1 18s rRNA primers. Bands produced were of the
desired length (~800bp) in all samples. Lane 1, SC A; Lane 3, SC
C; Lane 4, SC D; Lane 5, Sinularia sp.; Lane6, negative control;
Lane 7, 1kb Plus DNA ladder.

Intact GFP-like gene fragments are found in Soft corals A, C, and D

Primers used to screen soft corals SC A, SC C, and SC D for GFP

gene homogues were SC 11 FP ORF F which was originally designed by

Concepcion (2008) for amplification of the open reading frame of Alcyonium

sp.and SC 12 FP ORF R which was originally designed by Yunque (2007)

based on an internal sequence of Sarcophyton sp. In Fig 10, it is shown that

the three soft coral libraries yielded intact GFP-like gene fragments of the

desired length (~650 bp). Sequencing is still needed though to confirm identity

of the fragments.

22
Figure 10. Amplification of GFP-like gene fragments from three
soft coral samples SC A, SC C, and SC D. Bands produced were of
the desired length (~>650bp) in all samples. Lane 1, SC C; Lane 2, SC
D; Lane 3, SC A; Lane 5, negative control; Lane 6, 1kb Plus DNA
ladder.

23
Successful amplification of a >650bp fragment from Sinularia sp.

Using the primer pair SC1 ORF F and SC12 FP ORF R, a presumed

full-length GFP gene of Sinularia sp. was successfully amplified through

Touchdown PCR after optimization of the PCR profile, yielding a band just

above 650bp mark of the molecular weight ladder (Figure 11). Alignment and

editing of the longest contiguous sequences provided for Sinularia sp.

obtained from a previous study by Nabor (2009) to know the theoretical

complete sequence of Sinularia sp. GFP gene homolgue shows that it has a

GFP gene fragment 678bp in size. Sequencing is still needed though for

confirmation.

Figure 11. Full length GFP gene amplification of Sinularia sp. The
bands produced were of the desired length (~>650bp). Lane 1, SC A;
Lane 3, Sinularia sp.end-end GFP fragment.
24
Novel GFP-like gene successfully ligated into a bacterial expression
vector

With the assumption that the full-length GFP-like gene of Sinularia sp.

has been amplified, ligation into bacterial expression vector was done for

subsequent protein expression upon transformation. In Figure 12, a band of

length of about 850bp was produced upon amplification of the TOPO cloning

reaction using the vector specific T7 forward primer and gene specific SC 12

FP ORF R primer. This was the desired length since the theoretical size of

Sinularia sp. is 678bp while the distance of the T7 priming site to the cloning

site is 146bp, resulting to theoretically 824bp of product. For this ligation to be

important though, transformation of E.coli using this plasmid should be next

done.

Figure 12. Successful ligation of Sinularia sp.

full-length GFP-like gene amplification PCR
product into pEXP5-NT/TOPO bacterial
expression vector. A band about 850 bp was
produced using the primers T7 forward and SC12
ORF R.

25
26
 CONCLUSIONS

Whole-mount fluorescent images of three new soft coral samples, SC

A, SC C, and SC D were taken to confirm presence of fluorescent parts

suggested by their brightly colored tentacles. Screening for GFP-gene like

sequences using the intact cDNA libraries constructed yielded bands of

expected length for all three samples.

For Sinularia sp., a forward primer was designed and used along with a

previously designed reverse primer to amplify the full-length GFP gene of the

soft coral using an optimized Touchdown PCR profile. This fragment was then

successfully ligated into a bacterial expression vector, ready for

transformation of E.coli and protein expression.

27
 RECOMMENDATIONS

For molecular identification of the soft corals, the 18s rRNA amplified

should be sequenced. Similarly, the GFP-gene like fragments should also be

sequenced to confirm if the amplified products belong to the GFP gene

superfamily. Further RACE-PCR experiments should then be performed. The

ligated reaction product containing the full gene sequence of Sinularia sp, on

the other hand, should be used in the transformation of E.coli for subsequent

protein expression of this novel GFP gene

28
 LITERATURE CITED

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March 2011 from http://www.scribd.com/doc/14336575/farming-soft-
corals

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USA: Springer.

Goldys, 2009. Fluorescence applications in biotechnology and life sciences.

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Hinterdorfer, P. and Oijen, A. 2009. Handbook of single-molecular biophysics.

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species. Nat. Biotechnol. 17, 969-973.

Nabor, M. 2009. Full length cDNA sequencing, phylogenetic analysis, and

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Sanders J. and S. Jackson. 2009. The discovery and development of the

green fluorescent protein, GFP. Chem. Soc. Rev. 38, 2821-2822.

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Smith, C. 2007. Keeping tabs on fluorescent tags. Nature Methods. 4. 755-
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Verkhusha, VV & Lukyanov, KA. 2004. The molecular properties and

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Yang et al. 1996. The molecular structure of green fluorescent protein. Nat.
Biotechnol. 14, 1246-1251.

Yunque, A. 2007. Green fluorescent protein (GFP) gene homologues from

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30
 APPENDICES

Appendix A

PCR Profile
Primer Combination: 18s rRNA F1/R1
Samples: SC A, C, D, 1

Hold 94˚C 3 mins

94˚C 1 min
30 cycles 55˚C 1 min
72˚C 1 min

Hold 72˚C 7 mins

Hold 4˚C ∞

31
Appendix B

Touchdown PCR Profile

Primer Combination: SC 11 FP ORF F/ SC12 ORF R
Samples: SC A, C, D

Hold 94˚C 2 mins

15 cycles 94˚C 30 seconds

-1˚C/cycle 58.3˚C 45 seconds
72˚C 45 seconds

94˚C 30 seconds
25 cycles 45.3˚C 45 seconds
72˚C 45 seconds

Hold 72˚C 7 mins

Hold 4˚C ∞

32
Appendix C

Touchdown PCR Profile (End-to-end)

Primer Combination: SC 1 ORF F/ SC12 ORF R
Samples: 1

Hold 94˚C 2 mins

15 cycles 94˚C 30 seconds

-1˚C/cycle 68.0˚C 45 seconds
72˚C 45 seconds

94˚C 30 seconds
25 cycles 70.3˚C 45 seconds
72˚C 45 seconds

Hold 72˚C 7 mins

Hold 4˚C ∞

33
Appendix C

Confirmation of ligation
Primer Combination: T7 forward/ SC12 FP ORF R

Hold 94˚C 10 mins

94˚C 1 min
25 cycles 55˚C 1 min
72˚C 1 min

Hold 72˚C 7 mins

Hold 4˚C ∞

34
Appendix D

Primer Sequences

SC1 ORF F 5’ ATG AGC GTG ATA AAG CAG GAG ATG 3’
SC11 FP ORF F
5' ATG AGT GTG ATT AAA CAA GAA ATG AAG A 3'
(Concepcion, 2008)
SC12 FP ORF R
5’ TTA CTT GGC TTT ACT TGG CAG 3’
(Yunque, 2007)
T7 forward 5' TAA TAC GAC TCA CTA TAG GG 3'

35
Appendix E

Figure 13. TOPO® Cloning Site of pEXP5-NT/TOPO®

36
Appendix F

Figure 14. Alignment of Sinularia sp. (SC1) complete ORF sequence to

the primers used.

37