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Materials and Methods 2

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by

Blue Flower

on 30 June 2013

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Transcript of Materials and Methods 2

Materials and Methods
Reverse
Transcription

PCR
High-fidelity Pfu DNA polymerase was used to amplify S. mansoni genes to avoid errors that may lead to the expression of a faulty protein upon induction of protein expression.
Primers 16 and 17
gave 2 bands in
the agarose gel
Gel Extraction
using GeneJET
Gel Extraction Kit
cDNA was used as a PCR template
Cloning
T4 DNA Ligase
Transformation
into the appropriate
host
pTX8048
For the purpose of:
pNZ8048
pQE31
For the expression of schistosomal antigens in L. lactis NZ9000
Sequencing
Protein Expression
All PCR amplified genes were sent for automated DNA
sequencing
by GATC Biotech sequencing services (Germany) once using their forward and another time using their reverse primers.
E. coli M15 (pREP4) strains
IPTG
L. lactis NZ9000
strains
Nisin
Induction of Protein Expression
from culture supernatants of the nisin-secreting strain L. lactis NZ9700
His-tagged Protein
Purification of Proteins
using Ni-NTA metal-affinity
Chromatography
under native conditions
under denaturing conditions
For:
L.lactis NZ9000 strains
E. coli M15 (pREP4) strains
For:
E. coli M15 (pREP4) strains
1. Lysis of bacterial cells
By beating the cells in a Micro-MiniBeadbeater with glass beads for 3 one-minute beats with one-minute pauses on ice in between.
By stirring of the cells with denaturing lysis buffer containing 8 M urea for 1 hour at room temperature.
2. Protein binding to Ni-NTA
Bacterial strain containing
the plasmid and insert
3. Wash
By passing
the lysate
through
the column
By stirring
of the lysate
with the resin
for one hour
using wash buffer
containing a low
concentration of imidazole
by lowering the
pH to 6.3
4. Elution
using high concentration
of imidazole to
replace the bound
histidine residues
by lowering the
pH to 4.5
His-tagged protein in its native soluble form
Denatured His-tagged protein
expression of schistosomal antigens in L. lactis NZ9000 fused to E. coli ThioredoxinA protein
PCR amplification of His-tagged genes for insertion into pNZ8048
Construction of gene fusions
For the expression of schistosomal antigens in E. coli M15 (pREP4)
Extraction
Purification of Poly A mRNA
To obtain an
mRNA-enriched template for successful downstream RT-PCR
Liver Perfusion
S. mansoni-infected
hamster from
TBRI, Guiza
Adult worms
Collection of
adult worms
BIOZOL
total RNA
extraction reagent
Total RNA
Extraction
By treatment
with DNase I
to avoid
amplification
of the genes
with the
intervening
introns
DNase I was removed using RNA Purification Kit that utilizes
a spin-column technology
Removal of
Genomic DNA
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Western Blot
Coomassie Blue
staining
Protein Quantification by Bradford Assay
Bradford assay depends on a dye color change from brown to blue that is proportional to the protein concentration
BSA Standard curve was plotted
and total yields of proteins produced were calculated
S. mansoni cDNA containing
reverse transcripts of the genes of interest
Poly A mRNA containing
mRNA transcripts
of the genes of interest
All resulting sequences were searched for homology against GenBank database using
Nucleotide BLAST
(BLASTN) search tool.
All sequences were
aligned
with the sequences published in the NCBI nucleotide database using Lasergene DNA Star software.
denaturing
native
RNA
Full transcript