BTE 204 ASSIGNMENT

Name: Sadman Rashid Abir

ID: 20136004

SEC: 01
Reporter genes are used as indicators for gene expression of a desired gene. Reporter genes
produce proteins that can be easily detected and measured. These genes are when introduced
into the target cells produce receptors for proteins that binds an injected probe. Reporter genes
are inserted into the regulatory region of a gene of interest that is usually responsible for a
systematic expression of a particular gene. Transcription of the reporter genes are controlled by
these regulatory regions which could be a promoter region too. This way by injecting a reporter
gene whose regulatory region is tissue specific we can closely monitor the behaviour of gene
expression, also regulatory region which are cell specificity keeps the gene expression
restricted to cells . Reporter genes should be non toxic to cells, their assays should be sensitive
and reliable. Reporter genes can be classified into two parts (i) non fluorescent genes (ii)
fluorescent genes. There are also some random reporter strains that will be discussed along
with reporter genes in this topic.

CAT:

Chloramphenicol acetyltransferase (CAT) is an example of non fluorescent reporter genes. CAT

was the first reporter gene that monitored the transcriptional activity of the cell. CAT is a
bacterial enzyme. Chloramphenicol is an inhibitor of prokaryotic protein synthesis. This inhibitor
can be detoxified using chloramphenicol acetyltransferase. Acetyl group from acetyl Co A is
transferred the 3 hydroxyl group of chloramphenicol.Bacterial cells growth is affected by
chloramphenicol ( CAM). CAM inhibits protein synthesis by preventing the formation of peptide
bonds thus bacterial growth is inhibited. We will discuss how CAT works.

First the promoter of our gene of interest is to be removed. Then this promoter is to be cloned
into the into multiple cloning site of Vector downstream of a CAT gene as a result CAT gene will
now be under control of our desired genes promoter. Then we use a step called transfection to
insert our vector into cells of eukaryotes. AFter transfection the cells containing the vectors
would transcribe and translate to produce the CAT enzyme. Then we will harvest the proteins
produced by these cells which will contain CAT enzymes along with other proteins produced by
the cells. Then we can observe the reaction that the CAT enzyme catalyzes. We know CAT
enzyme is acetyl transferase that means it will take acetyl group from acetyl CoA and transfer it
to CAM or chloramphenicol. After the reaction we would be able to see acetylated 14C-CAM
also unacetylated 14C- CAM, Free and bound CAT enzyme, excess acetyl CoA. Here
acetylated CAM is our focus because this is where the CAT enzyme catalyzed. Now to separate
acetylated CAM from unacetylated CAM we use Thin layer chromatography. Here sample are
placed on the bottom of the plate called origin and ten we will dip the bottom of the plate in a
mobile phase solvent and as a result all the components will be separated based on their
polarity. Autoradiography is used to visualize the 14C-CAM. Unacetylated 14C CAM remains at
the bottom of the plate and Acetylated 14C-CAM remains at the top of the plate
Infection caused by Agrobacterium tumefaciens in dicotyledonous plants causes neoplastic
growth. A part of agrobacterium Ti plasmid, T DNA is integrated into the chromosome of
infected plants which is responsible for encoding opine synthesizing enzymes. Opines are
amino acid found only in plans. They are often formed in plant tissue after feeding of arginine
which is used as precursor for opine synthase. AMong the opines the notables are nopaline and
octopine. Nopaline is formed by direct condensation of arginine and alpha ketoglutaric acid and
octopine is formed due to the condensation of between arginine and pyruvic acid. Opines
perform as the carbon source for the invading agrobacterium . MAny oncogenic strains of the
agrobacterium induce tumor which synthesizes opine because these can be catabolized easily
by bacteria. Thus bacterial genes can be expressed in the transformed cell as opine cannot be
detected in normal tissue. We will discuss the opine synthase assay.

Opine Synthase:

Seeds of soybean and tobacco were sterilized with a 5% solution of NaOCl and germinated
agar. Callus was separated from the hypotycol of sterile seedling. These callus was then
incubated with proper opine precursors like arginine at concentrations of 100mM and puruvate
along with NADH for ocs and with alpha ketoglutaric acid and NADH for nos. Arginine enriched
callus would grow for 3-5 days. These samples were analysed using paper electrophoresis
suing two buffer system. One of the buffer is composed of acetic acid and water and
electrophoresis is conducted for 1.5 h at 400 v. Another buffer is composed of 5% pyridine and
0.25% aqueous solution of acetic acid. Electrophoresis is conducted for 1.5 h at 350V. After
electrophoresis the paper was dried and phenanthrenequinone positive spots were visualized
and octopines and nopalines were separated from the solvent.

SEAP:

Secreted alkaline phosphatase has become a promising tool for investigating promoter activity
in transfecting eukaryotic cells . The SEAP reporter gene encodes a truncated form of the
human placental alkaline phosphatase gene that lacks the feature to anchor to the membrane.
Thus the protein can be easily excreted from the transfected cells without causing cell lysis.
SEAP assay utilizes enzyme activity of alkaline phosphatase to dephosphorylate the
chemiluminescent alkaline phosphatase substrate CSPD into an unstable dioxetane anion
which emits light upon decomposition .

For performing SEAP assay first the media is to be removed from the cultured cells that have
been transfected with seap reporter vector. Assay reagents should be diluted with chemicals of
appropriate concentration and allow to equilibrate at room temperature. Sample should be
diluted with a dilution buffer and should be incubated for 30 min at 65C.The dilution buffer
contains inhibitors that target non placental alkaline phosphatase Then add assay buffer to each
sample and incubate for 5 min at room temperature.. Then add substrate reagent and incubate
for 10- 20 min. Once the reagent is added light output reaches a stable level after some time .
After that measure light output using either a single tube or microplate luminometer with a .1
to.5 integration time.
phoA:

Alkaline phosphatase gene( phoA) can be used as a reporter gene in mycoplasmas. PRomoter
region for the elongation factor (ltuf) and signal acylation sequence ulhA 1.1 gene from
mycoplasma and also the encoding region of phoA were assembled in a transpoon containing
plasmid (pTAP).

The phoA gene from E. coli was cloned and fused to the vlh A1.1 lipoprotein acylation signal
sequence [28], the first 5 residues of the mature protein, under the guidance of the ltuf promoter
and cloned into transposon containing plasmid (pTAP). Another plasmid (pTP) lacking the signal
and acylation sequence was also developed . Both the plasmids were introduced into the E
coliD5a by electroporation using gene pulser. After ecombiantion clones were selected and
cultured and plasmid DNA were extracted. These transformed cells were incubated at 37C.
Then 500 microlitre aliquot was inoculated into the medium. To Detect alkaline phosphatase
activity in Transformed mycoplasmas a single tablet of nitro blue tetrazolium and was dissolved
nad sprayed into the colony. After some time the colonies would emit blue color as an indication
of PhoA gene. These way phoA gene can be used to observe transformed mycoplasmas
xylE:

xylE gene isa promoterless gene of Pseudomona Putida. xylE gene converts colorless catechol
to yellow hydroxymuconic semialdehyde. xylE gene was placed under a promoter named galP1.
This promoter is glucose repressed and galactose induced. After being cultured on agar plates
,colonies of bacteria on galactose turned bright yellow and colonies on glucose remained white.

To determine whether this reaction could be used to detect galP1 driven expression of xylE. S.
lividans containing pXE3 or pXE4 were replicated on agar plates containing either glucose or
galactose as their carbon source. Then after spraying 0.5M solution of catechol glucose
containing pXE3 cells remained white but pxE# containing galactose turned bright yellow.
This activity of xylE and galp 1 fusion was measured in crude cell extracts with a simple
colorimetric assay. Colonies growing in glucose shows no color and thus shows no exrepssion
of xylE gene
Besides these there are many more reporter genes that can be used to express different types
of functions. Reporter genes are a useful tool for genetic engineering and for decades scientists
have been finding new reporter genes for new experiments.