*PROJECT ON LUCIFERASE GENE CLONING 1. SUITABLE VECTOR 2. RESTRICTION ENZYMES ALONG WITH THEIR RESTRICTION SITES 3. CLONING PROCESS 4. EXPRESSION 5. PGL-2 CONTROL VECTOR DIAGRAM 6. COMPARISON OF VECTOR IN MORE THAN ONE HOST 7. NEW INNOVATIVE IDEA FOR CLONING 8. CONCLUSION 9. SUMMARY

1. PROJECT ON
LUCIFERASE GENE
CLONING
PRESENTED BY: AYESHA KABEER
UNIVERSITY OF SIALKOT

2. Luciferase gene
 A gene obtained from firefly Photinus pyralis.
 Luciferase gene encodes luciferase enzyme
which binds to a chemical present in cell called
luciferin and convert it into oxyluciferin through
the process of oxidation with the release of light.
• Luciferin + O2 Oxyluciferin
Luciferase enzyme

3. Luciferase gene
• Suitable vector:
• The vector which is suitable for the cloning of luc
gene is pGL2-Control Vector.
• Restriction enzymes along with their restriction sites
HindIII 5’…AAGCTT…3’
3’…TTCGAA…5’
BamHI 5’…GGATCC…3’
3’…CCTAGG…5’
SaII 5’…GTCGAC…3’
3’…CAGCTG…5’

4. Restriction enzymes along with
their restriction sites
XhoI 5’…CTCGAG…3’
3’…GAGCTC…5’
BgIII 5’…AGATCT…3’
3’…TCTAGA…5’
KpnI 5’…GGTACC…3’
3’…CCATGG…5’
SacI 5’…GAGCTC…3’
3’…CTCGAG…5’
MluI 5’…ACGCGT…3’
3’…TGCGCA…5’
NheI 5’…GCTAGC…3’
3’…CGATCG…5’

5. Cloning process
 Before cloning, firstly the pGL2-Control Vector is
designed in which the luc gene is present.
 Then the cells of E.coli JM109 are transfected
with this pGL2-Control Vector and then cloned.
 The transfection of vector into E. coli may be
mediated by calcium phosphate, or
electroporation.

6. Expression
 pGL2-Control Vector contains the SV40 promoter
and enhancer sequences, resulting in strong luc
expression in cells.
 This plasmid is useful in monitoring transfection
efficiency.

7. pGL2-Control Vector

8. Comparison of expression in
more than one host
 The expression of luciferase gene in E.coli JM109
cells is greater than its expression in mammalian
cells as it is easy to handle E.coli JM109
compared to mammalian cells.
 Moreover, E.coli JM109 completes its one cycle
of replication in just twenty minutes and due this
we can get many gene clones and expressions in
one day.

9. New innovative idea for
cloning
 We can also clone luciferase gene by transfecting
E.coli cells with shuttle vector instead of pGL2-
Control Vector.
 Yeast Integrating Plasmid (YIp5) is an example of
shuttle vector.
Advantage of YIp5 shuttle vector is that it is
small in size (i.e. 5541 bp) as compared to pGL2-
Control Vector (i.e. 6047 bp) and due to this it is
more stable.
 Other example of shuttle vector is Yeast
Centromere Plasmid (YCp50).

10. Conclusion
It is concluded that luciferase gene can also be
cloned by the using shuttle vectors and its
expression can be obtained in both E. coli and
mammalian cells but greater in E. coli.

11. Summary
• Luciferase gene encodes luciferase enzyme which
catalyzes the oxidation of luciferin to oxyluciferin with
the release of light.
• Its cloning is done by transfecting E.coli JM109 cells
with a pGL2-Control Vector in which luciferase gene,
SV40 promoter, enhancer elements, ampicillin resistant
gene, origin of replication and restriction sites are
present.
• The transfection is done through the treatment of
electroporation or with calcium phosphate.
• The expression of luc gene in E. coli cells is greater than
in mammalian cells.
• The cloning of luc gene can also be performed by
transfecting E.coli cells with a shuttle vector i.e. YIp5
and YCp50.