This is to certify that this undergraduate thesis, entitled: “Fluorescence

imaging and construction of cDNA libraries for screening fluorescent

proteins from Philippine soft corals” and submitted by CECILE CHAN
DUNGOG to fulfill part of the requirements for the Degree of Bachelor of
Science in Molecular Biology and Biotechnology, is hereby endorsed.

____________________________
CYNTHIA P. SALOMA, Ph.D.
Thesis Adviser

The National Institute of Molecular Biology and Biotechnology accepts this

undergraduate thesis in partial fulfilment of the requirements for the Degree of
Bachelor of Science in Molecular Biology and Biotechnology.

____________________________
CYNTHIA T. HEDREYDA, Ph.D.
Director
National Institute of Molecular Biology
and Biotechnology (NIMBB-UPD)

ii
 ACKNOWLEDGEMENTS
HAHA!

iii
 ABSTRACT

With the growing applications of fluorescent proteins in live cell

imaging, several efforts through cloning and mutagenesis have been made to

find novel GFP-like proteins with desirable properties for biological imaging. In

this study, these efforts are extended to the diverse marine life in the

Philippine seas. Nonbioluminescent, brightly colored soft corals originally from

Batangas and Zambales were collected from Cartimar, Pasay and initially

screened for green fluorescent protein homologues through whole-mount

fluorescence imaging. Excitation with blue light revealed intense fluorescence

on localized regions of the soft corals. Total RNA of each sample was

exracted and used to construct intact cDNA libraries for further screening of

presence of GFP-gene homologues using primers specific to GFP genes of

other soft coral species. Optimization of PCR profile was also done for the

complete amplification of the GFP gene of Sinularia sp found to be 678bp in

size, yielding a fragment of the expected length. Sequencing of amplicons

produced should be done for verification of successful amplification of desired

genes, as well as transformation of E.coli using the ligated full-length GFP

gene sequence of Sinularia sp.into a bacterial expression vector for protein

expression.

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 TABLE OF CONTENTS
Page

Certification Page ii
Acknowledgements iii
Abstract vi
List of Tables viii
List of Figures ix

Introduction 1
Objectives 2
Significance of Study 2
Scope 2

Review of Related Literature

3
Bioluminescence vs. Fluorescence
3
Aequorea Victoria Fluorescent Protein
4
Structure
5
Fluorophore formation
7
Spectral properties
8
GFP variants
8
Other Members of the GFP Superfamily
8
Soft coral taxonomy and morphology
9
Diversity of spectral properties
11
Amino acid residues necessary for fluorescence
12
Current Applications of Fluorescent Proteins

Materials and Methods

14
Sample collection

14
Fluorescence imaging for initial screening

14
Total RNA extraction

16
cDNA library synthesis

16
Amplification of GFP homologue sequences

v
 16
Touchdown PCR

17
Large scale PCR

Wizard™ Prep DNA Purification System (Promega) 17

Gene insertion and ligation into pEXP5-NT/TOPO ® 18

Results and Discussion

Nonbioluminescent soft corals exhibit bright coloration 19

Fluoresce imaging confirms presence of fluorescent 19

parts under blue light (λEx = 450- 490nm)
Samples stored in RNAlater® yield total RNA ready for 21
cDNA library construction

Successful amplification of partial 18s rRNA fragments 22

of samples suggests construction of intact cDNA
libraries

Intact GFP-like gene fragments are found in soft corals 22

A, C, and D
Successful amplification of a >650bp fragment from 23
Sinularia sp.

Novel GFP-like gene successfully ligated into a 24

bacterial expression vector
Conclusions 26

Recommendations 27

Literature Cited 28

Appendices 30

vi
 LIST OF TABLES
Page
Table 1 Amino acid positions and residues critical for 11
fluorescent activity
Table 2 Nonbioluminescent parts with bright coloration of soft 21
coral samples exhibit green fluorescence

vii
 LIST OF FIGURES

Page
Fig 1 GFP structure highlights 5
Fig 2 General mechanism for GFP fluorophore maturation 6
Fig 3 Absorption and emission spectra of avGFP 7
Fig 4 Soft coral morphology schematic diagram 9

Fig 5 GFP-like proteins across the tree exhibit different spectral 10

properties
Fig 6 Nonbioluminescent soft coral samples with potential 19
GFP-like genes.
Fig 7 Fluorescence imaging reveals soft coral fluorescent parts 20

Fig 8 Total RNA extracts from dissected parts of soft corals 21

samples stored in RNAlater®
Fig 9 Amplification of partial 18s rRNA gene fragments of soft 22
coral samples SC A, SC B, SC C, and Sinularia sp. using
F1 and R1 18s rRNA primers.
Fig 10 Amplification of GFP-like gene fragments from three soft 23
coral samples SC A, SC C, and SC D.
Fig 11 Full length GFP gene amplification of Sinularia sp 24
Fig 12 Successful ligation of Sinularia sp. full-length GFP-like 25
gene amplification PCR product into pEXP5-NT/TOPO ®
bacterial expression vector

viii