Academia sinica IBC meeting 08/26

1. Gene cloning for APEX2 project to
understand protein-protein interaction
Speaker: Tzu-Hao Liu
Advisor: Dr. Chi-Kuang Yao
2019/08/26
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2. LRRK2 is a large, widely expressed, multi-
domain and multifunctional protein
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• LRRK2 is associated with Parkinson's disease.
• The most common mutant gene on LRRK2 is G2019S
• Disrupting kinase activity reduced mutant LRRK2 toxicity
by affecting LRRK2 levels rather than kinase activity itself.
• LRRK2 gene length is up to 10kb, it’s make me difficult to
transform.
LRRK2 protein
Ref: Li et al. Molecular Neurodegeneration 2014, 9:47
J Neurosci Skibinski G et al.(2014), 34:418–433.

3. Codons repeats in C9orf72 cause neuron toxic
• Expression of pure codon-repeated protein in Drosophila
caused neurodegeneration
• C9orf72 mutant in human was associated with ALS/FTD.
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https://ghr.nlm.nih.gov/gene/C9orf72#location
Gene Express in flies
Poly-GA100 Low toxic (Control)
Poly-GR100 Pathogenic
Poly-PR100 Pathogenic
Science. Sarah Mizielinska et al. (2014 September 5)

4. Drosophila eyes pattern and life time
difference between Poly-(PA/GA/GR/PR)
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Science. Sarah Mizielinska et al. (2014 September 5) Figure 3-A,B,D

5. We use APEX2 method to screen protein-
protein interaction
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Braden T. Lobingier et al.(2017)
We can know “when” and “where” protein-protein
Interaction happened via APEX2 method.

6. Construct plasmid by gene cloning technology
• Gene cloning is a method make us can recombine gene we interesting to another
gene fragments.
• Transform is a key step in gene cloning, using bacteria to select ideal plasmid.
• We use UAS-GAL4 system to express gene in transgenic flies.
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2012全國科展 謝庭軒 指導老師: 孫以瀚、吳雅嵐

7. Gene cloning progress report in this summer.
Draw gene map Primer design PCR fragments
DNA fragments
prepare
DNA fragments
ligation
TransformColony PCR test
Plasmid
purification
Restrictive
Enzyme test
DNA
sequencing
Finish
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LRRK2-wt
Poly-GR/PRPoly-GA
Tau-wt

8. Problems which I was encountered.
• Because of GGGGCC repeats, primer will bind to wrong site, I can’t get
sequence data of Poly-(GA).
• Poly-(PR) and Poly-(GR) also have 100 DPR codons repeats, thought I
have DNA sequence data, I can’t successfully create PCR fragments
because primer mis-binding.
• pUAST-LRRK2-APEX-HA is too large to construct, I can’t success
transform to DH5-alpha
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High efficiency transformation of Escherichia coli with plasmids, Hiroaki Inoue(1990)

9. 500 bp band gene in gel is a part of pUASTattB
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https://blast.ncbi.nlm.nih.gov/Blast.cgi

10. Change primer design strategy
Poly-(GR)excerpt Before Poly-(GR)excerpt After
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NotI
Change primers

11. So far, my discoveries
• In our situation, KpnI is more efficiency than KpnI-HF.
• Competent cell need 30 min recovery in LB broth.
• Some primers in pUAST /pUASTattB have multiple binding site.
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Heat shock time Incubation time Colony number
#LRRK2 3-1 42°C 90 sec 0 min 0
#LRRK2 3-2 42°C 50 sec 0 min 0
#LRRK2 4-1 42°C 90 sec 30 min 3
#LRRK2 4-2 42°C 90 sec 0 min 0

12. In our situation, KpnI is more efficiency than KpnI-HF
• In 37 °C
• Digest for 2 hours
• pUAST plasmid
• TAE-1%-agarose gel
• Kpn1 with buffer 2.1
• Kpn1-HF with cut smart
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uncutKpn1-HFKpn1 Experiment condition

13. SV40term-R is not a good primer when PCR
template is pUASTattB
PCR condition band at wrong site.
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100-200bp
1kb
• 98 °C for 3 min
• 98 °C for 10 sec
• 55 °C for 30 sec
• 72 °C for 60 sec
• 72 °C for 10 min
• 4 °C forever
35 cycles
pUASTattB-R
SV40term-R
So we redesign a new primer – pUASTattB-R

14. So far, my discoveries
• In our situation, KpnI is more efficiency than KpnI-HF.
• Competent cell need 30 min recovery in LB broth.
• Some primers in pUAST /pUASTattB have multiple binding site.
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Heat shock time Incubation time Colony number
#LRRK2 3-1 42°C 90 sec 0 min 0
#LRRK2 3-2 42°C 50 sec 0 min 0
#LRRK2 4-1 42°C 90 sec 30 min 3
#LRRK2 4-2 42°C 90 sec 0 min 0

15. In this summer internship, I learned a lot of
things.
• How to make agarose gel and run DNA electrophoresis.
• Knowledge and technology needed for gene cloning.
• How does DNA sequencing working.
• How to design primer for PCR and DNA sequencing.
• How to use restrictive enzyme and ligase.
• How to culture bacteria and simple sterile working.
• What is Gibson reaction and how to do it.
• How to transfer fruit fly and use CO2 to anesthetize fruit flies.
• How to pick larvae with phenotype we want.
• How to use fluorescence microscope.
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16. What can I do in future.
• Redesign primers to complete DPR fragments PCR.
• Confirm genomic DNA from fruit fly is complete and PCR must have
major band before doing further PCR reaction.
• Try another method to construct LRRK2-wt like ligation or
mutagenesis.
• Learn more knowledge about Drosophila and molecular biology.
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