In this PPT You can learn briefly about Reporter Gene and Gene fusion And Gene manipulation method. Reference From Microbiology. Author - Brock. 12th Edition,

1. • The Mandavi Education Society
T.Y.Bsc(Microbiology)
Krishna prajapati
Teaching Assistance(Microbiology)

2. Reporter gene and gene fusions

3. Introduction
• DNA manipulation has revolutionary the study of
gene regulation.
• A coading sequence from one source(the
reporter)may be fused to a regulatory region
from another source.
• Such gene fusions are often used in studying gene
regulation especially where assaying the level of
the natural gene product is difficult or time
consuming.
• They may also be used to increase expression of
desired gene product.

4. Reporter genes
• The key property of reporter gene
is that it encodes a protein that is
easy to detect and assay.
• Reporter gene are used for
variety of purposes.
• They may used to report on the
presence or absence of particular
genetic elements(such as
plasmid)or DNA inserted within
the vector.
• They can also fused to other
genes or to promoter of other
genes so that gene expression
can be studied.

5. LacZ gene
• The first gene to be used
widely as reporter gene was
lacZ, which encodes the
enzyme β-galactosidase.
• Cells expressing β-
galactosidasecan be
detected by easily by their
color on indicater plates
that contain the artificial
substrates Xgal ,which
cleaved by β-galactosidase
to yield blue colour.

6. Blue white screening .

7. Conti..

8. Green flourescent protein(GFP)
• Another widely used
reporter gene encodes
luciferase.
• This enzymes makes cells
expressing it luminescent.
colonies containing the
reporter system can be
detected by on agar plate
by their luminscense
against a large
background of other
colonies.

9. Conti..
• However,the expression of luciferase depends on
more than one gene beacause several accesary
factors are needed.
• By contrast, the green fluroscent protein needs
no accessary factors and is widely used as
reporter
• Gene for was GFP was originally cloned from the
jelly Aequorea victoria,the GFP protein may be
expressed in most bacterial cell.
• It is stable and causes little or no disruption of
host cell metabolism.

10. Conti…
• If expression of cloned
gene linked to that of GFP,
expression of GFP signals
that the cloned gene has
also been expressed.
• Recent advance in
fluorescent labeling now
allow the simultaneous
use of multiple
fluorescent markers.

11. Some like that..

12. Gene fusion..
• It is possible to
engineering construct
that consist of segment
from two different
genes.
• such construct are
known as gene fusions.

13. Conti..
• If the promoter that
controls a coding
sequence is removed, the
coding sequence can be
fused to different
regularly region to place
the gene under the
control of different
promoter alternatively.
• The promoter region can
be fused to gene whose
product is east to assay.

14. There are two types of gene fusion
Fig..
.
Operon fusion
• In operon fusion, a coading
sequence that retain its
own translation start site
and signals is fused to the
transcriptional signals of
another gene.

15. Gene fusion
Protein fusion
• In protein fusion, two
coading sequence are fused
with the result that they
share the same
transcriptional and
traslational start sites and
signals.
Fig…

16. Conti….
• Gene fusion are often used in
studying gene regulation, especially if
measuring the level of natural gene
product is difficult and time
consuming.
• The reulatery region of the gene of
interest is fused to coding sequence
for reporter gene, such as β-
galactosidase or GFP.
• The reporter is then made under the
condition that would trigger
expression of the target gene.
• The expression of reporter is assay
under variety of condition to
determine how the gene of interest is
regulated, where as protein fusion
reveal translational cantrol.

17. Reporter construction

18. Conti..
• Gene fusion may also be used
to test for the effect of
regulatory genes.
• Mutation that effect
regulatory genes are
inroducted into cells carrying
gene fusion, and expression is
measured and compared to
cell lacking the regulatory
mutation.
• This allows the rapid screening
of multiple regulatory genes
that are suspected of
controlling the target gene.