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TET system in Aspergillus nidulansTranscriptome update

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The first tet-regulated gene expression system for use in mammalian cells was ... Ligate into Sma1 site of PPl6. Tet fragment, gel extracted and purified. ... – PowerPoint PPT presentation

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Title: TET system in Aspergillus nidulansTranscriptome update


1
TET system in Aspergillus nidulans/Transcriptome
update
  • Lesley Lockhart
  • University of Sheffield

2
Tetracycline regulated inducible gene expression
The first tet-regulated gene expression system
for use in mammalian cells was developed by
Gossen and Bujard. Tetracycline system based on
the Tn10 tetracycline-resistance operon of
E.coli. Expression of tetracycline resistance
genes is repressed by the binding of a
tetracycline repressor (tetR) to operator
sequences (tetO). In the presence of
tetracycline, the repressor is inhibited from
binding to the operator and transcription is
derepressed.
3
Yeast Tet system Previously tested in A.
nidulans (Caroline Foster). tetR fused to Gal4
activation domain to create transactivation
protein tTA, which can bind to the tetO sequences
in the absence of tetracycline , or is repressed
in the presence of tetracycline (tet
Off). Laccase placed under control of 7 tandem
repeats tet O and the HOP1 mini promoter
allowing it be repressed in the presence of
tetracycline. No activity of laccase seen
P
tetR
Gal4
Dox
Tet Off
- Dox
tet O
tet O
Laccase
HOP1
HOP1
Laccase
No expression
Expression
4
Mammalian TET system A transactivator protein
tTA, created by a fusion of tetR with the
transcriptional activation domain of the VP16
protein A gene of interest can be placed under
the control of a modified promoter with multiple
copies (7) of tetO and a human CMV minimal
promoter. Thus allowing it be repressed in the
presence of tetracycline.
P
Tet Off
TetR
VP16
Dox
- Dox
Tet O
Tet O
Laccase
CMV
CMV
Laccase
Expression
No expression
5
Cut with Hpa1
Tet fragment, gel extracted and purified.
Ligate into Sma1 site of PPl6
6
Transformed plasmid into strain
JK3?acuA. Transformants were checked by Southern
hybridisation
7
Plasmid linearised with Apa1 was used as a probe
8
Wild type JK3 acuA
PPL6-TETA T17
PPL6-TETA T20
PPL6-TETA T21
PPL6-TETA T23
PPL6-TETA T27
Hyperladder 1
Hyperladder 1
Hyperladder 1
10,000 bp
8,000 bp
Homologous integration of plasmid
6,000 bp
5,000 bp
4,000 bp
Wild type band
9
(No Transcript)
10
Xba1 (111213)
Xba1 (108126)
3087bp
Probe Plasmid cut with HindIII and EcoRV to
release a band containing ArgB
109243
110436
ArgB
3082
4272
ArgB
0
MP158 6937bp
Xba1 (114310)
Xba1 (108126)
Xba1 (1209)
Xba1 (118150)
TET
117373
109243
110436
116183
ArgB
ArgB
TET
3840 bp
6184 bp
11
MP158 T2
Hyperladder1
MP158 T4
MP158 T7
Wild type
MP158 T5
MP158 T6
MP158 T1
MP158 T3
MP158 T8
Partially digested DNA
10,000 bp
8,000 bp
6,000 bp
5,000 bp
Correction integration of plasmid
4,000 bp
3,000 bp
2,000 bp
Wild type band
12
Laccase assay- Transformants were stabbed on to
plates containing ABTS (2,2-Azino-bis(3-ethylbenz
thiazoline-6-sulfonic acid)) with and without
doxycycline Transformants producing laccase
should turn the ABTS on the plate around them
green A control spot of laccase was stabbed onto
plate
Wild type control strain
Control
13
Wild type strain R153 (hbrB) and hyperbranching
mutant hbrB3 were grown in a steady state
continuous culture at temperatures 42oC and
30oC. RNA extracted from strains R153 and hbrB,
labelled with Cy5 or Cy3 and hybridised against
microarray slides. Experiment 1 Differences in
gene expression of R153 at 42oC and hbrB at 42oC.
(Biological repeats of these slides
unfortunately, were R153 at 42 v. hbrB3 at 40oC
due to technical difficulties). Experiment 2
Differences in gene expression of R153 at 30oC
and Hbr B at 30oC. Hypothesis Genes up or down
regulated in the hbrB3 strain at 42oC but not at
30oC may be linked to hyperbranching phenotype.
14
Data Analysis
Data was normalised using MaxD Intensity
Dependent Normalisation. Data was filtered to
remove spots that had been flagged as bad by the
scanning software. Log ratios of normalised data
Cy5/Cy3 . Statistical analysis was carried out
using Gene Spring.
15
Up regulated genes in the HBRB3 strain at 42oC.
16
Down regulated spots in HBRB3 at 42oC
17
What next?
Is the data repeatable? Repeat of fermentations
to check reproducibility with both strains at
40 -Northern analysis to confirm
results -identify unknown genes on
array? -whole genome arrays? Decide which genes
to focus on possibly AN5781
AN0287 AN2760
18
Acknowledgements
Geoff Turner Caroline Foster Manda Gent Karin
Lanthaler E28
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