Gs Bacillus

Name________________________

StarBiochem




Green
Fluorescent
Protein
(GFP)
Exercise





Learning
Objectives

In
this
exercise,
you
will
use
the
StarBiochem
and
StarORF
software
tools
to
explore:

• the
sequence
of
the
GFP
gene
and
the
structure
of
its
protein

• the
fluorophore
that
accounts
for
GFP’s
fluorescence




Background

GFP
is
a
fluorescent
protein
isolated
from
several
organisms
including
the
pacific
jellyfish
Aequoria
Victoria.

GFP

converts
two
specific
wavelengths
of
blue
light
into
green
fluorescent
light
by
energy
transfer.




Proteins
that
fluoresce
contain
components
called
fluorophores.

In
GFP
the
fluorophore
originates
from
an
internal
tripeptide
sequence
(Ser‐Tyr‐Gly)
which
is
post‐translationally
modified
to
the
structure
shown
below,
a

4‐(p‐hydroxybenzylidene)‐imidazolidin‐5‐one.
















It
is
worth
noting
that
the
formation
of
GFP’s
fluorophore
is
an
autocatalytic
process
that
requires
no
cofactor
or

enzymatic
reaction.

This
fluorophore
is
highly
stable
even
at
high
temperatures
and
various
pH
conditions.



The
gene
for
GFP
has
been
isolated
and
has
become
a
useful
tool
for
making
fusion
proteins
in
which
GFP
is
linked
to
other
proteins
and
functions
as
a
fluorescent
protein
tag.

GFP
tagging
can
be
used
in
a
wide
range
of

applications:
as
a
tracer
for
cell
lineage,
as
a
reporter
of
gene
expression,
or
as
a
reporter
of
protein‐protein

interactions.




Getting
started
with
StarORF

StarORF
is
a
six‐frame
translation
software
tool
for
exploring
gene
sequences.

•To
get
to
StarORF,
please
navigate
to
http://web.mit.edu/star/ORF.

• Click
on
the
Start
button
to
launch
the
application.

• Click
Trust
when
a
prompt
appears
asking
you
if
you
trust
the
certificate.

• In
the
Input
box,
paste
the
sequence
provided
below.


Ver.
5
‐
D.
Sinha
and
L.
Alemán

1

Name________________________

StarBiochem



The
cDNA
sequence
of
the
GFP
gene
(5'→
3'
direction)
is
shown
below.



tacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgatgttaatgggc
aaaaattctctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgggaagctacctgttccatggc
caacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcga
gttaaaaggtattgattttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataatgtatacatcatggcagacaaac
caaagaatggaatcaaagttaacttcaaaattagacacaacattaaagatggaagcgttcaattagcagaccattatcaacaaaatactccaattgg
cgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaacaattactaaat
tctcagggttcctggttaaattcaggctgagactttatttatatatttatagattcattaaaattttatgaataatttattgatgttattaataggggctattt
tcttattaaataggctactggagtgtat



Using
the
StarORF
software
tool
answer
the
following
questions.



1

What
is
the
length
of
the
GFP
cDNA?

Answer






2

What
is
the
percentage
of
each
nucleotide
base
in
the
GFP
cDNA?

Answer






3

What
is
the
sequence
of
the
first
ten
bases
of
GFP’s
noncoding
DNA
strand
(5'→ 
3'
direction).

Answer






4

What
is
the
sequence
of
the
first
15
bases
of
GFP’s
mRNA
(5'→ 
3'
direction)?

Answer






5

Using
the
cDNA
sequence
provided
in
this
exercise,
you
estimate
GFP’s
mRNA
length.

In
your
laboratory,
you
then
isolate
total
GFP
RNA
from
jellyfish
and
resolve
it
on
a
gel
based
on
the
RNA
size
difference.

You
find
two

different
GFP
RNAs:
one
of
the
RNAs
is
bigger
than
your
estimate
and
the
other
RNA
is
the
same
size
as
your

estimate.

How
do
you
explain
this
result?










Ver.
5
‐
D.
Sinha
and
L.
Alemán

2

Name________________________

StarBiochem



Answer














...