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Team:UCSF/Cathy Liu Notebook
From 2008.igem.org
Objective:
Connect silencing/antisilencing to two distinct biological functions (aka "memory readouts")-
1. Cellular differentiation- filamentous growth
2. "Immune response"- antimicrobial peptide secretion
Rationale:
Demonstrate the modularity of the chromatin bit i.e. that it can be used with multiple outputs. Demonstrate that silencing or antisilencing can initiate differentiation.
Milestones:
1. Connect silencing/antisilencing to the filamentation pathway ("differentiation"):
a. Check if deletion of DIG1 signaling leads to filamentation.
b. Demonstrate that silencing of DIG1 leads to filamentation.
2. Connect silencing/antisilencing to antimicrobial peptide secretion by yeast
a. Research literature for antibacterial peptides
b. Make sure peptides can be secreted by yeast and determine which strains are viable
c. Test yeast supernatant effects on bacterial growth
The Road Towards Success
WEEK 1: 6/23/08-6/29/08
[Antisilencing work] PCR Lex A NLS Ligate into Topo; transform Mini prep Topo colonies Test digest with EcoRI
Mini prep FRE (Leu) colonies
Mini prep FRE (Ura) colonies
Design primers for Dig1 knockout
WEEK 2: 6/30/08-7/6/08
Sequence Lex A NLS-Topo
TEC1* (T273M) transformation
Mini prep T273M colonies
Double digest T273M with PstI and KpnI
Double digest pRS304 vector with PstI and KpnI
Run gel; gel purify
Ligate; transform
Religate digests; transform
Redo pRS304-T273M double digest with PstI and KpnI
Run gel
Sequence FRE plasmids
Double digest FRE plasmids with BamHI and ClaI, SacI, EcoRI, or EcoRV
Run diagnostic gel
Redigest FRE plasmids
PCR Dig1 fragments
Replica plate Lex A-Sir 2-Dig1KO (Nat)
Replica plate Lex A-Sir 2-Dig1KO (HygB)
Replica plate CB008-Dig!KO (Nat)
Replica plate CB008-Dig1KO (HygB)
WEEK 3: 7/7/08-7/13/08
Design oligos to check Dig1KO strains
Yeast colony PCR
Run diagnostic gel- inconclusive
RePCR Dig1
Digest pRS304-T273M with PstI
Digest pRS304-T273M with KpnI
Run diagnostic gel- unsuccessful
Mini prep more pRS304 vector
Run gel; gel purify
Ligate pRS304 and T373M; transform
Mini prep colonies
Digest plasmid; run diagnostic gel- T273M not visible
(Redo mini preps...)
Redigest pRS304-T273M with PstI
Redigest pRS304-T273M with KpnI
Run gel; gel purify
Boil Lex A 8x operatos
Ligate with PstI cut pRS304-T273M; transform
Ligate with KpnI cut pRS304-T273M; transform
Overlap PCR Cecropin A antimicrobial peptide
Overlap PCR LL-37 antimicrobial peptide
Overlap PCR Sarcotoxin A antimicrobial peptide
Run gel; gel purify
Clone alpha factor signal sequence into Topo vector
WEEK 4: 7/14/08-7/20/08
Redigest pRS304-T273M with PstI
Run gel; gel purify
Ligate with PstI cut pRS304-T273M; transform
PCR (PstI) Lex A-pRS304-T273M
Run diagnostic gel
Redo ligation; redo transformation
Remake Lex A operators
Digest Lex A with PstI
Digest Lex A with KpnI
Run gel; gel purify
Conduct Western Blot to check Dig1KO strains
Dig1KO unsuccessful
Mini prep Adh1p vector
Mini prep Cyc1p vector
Digest Adh1p vector with Aar1
Digest Cyc1p vector with Aar1
Run gel; gel purify
Redigest Cyc1p vector with Aar1
Run gel; gel purify
Digest AFSS-Topo with Aar1
Run gel; gel purify- unsuccessful
Remake AFSS-Topo
WEEK 5: 7/21/08-7/27/08
Topo clone AFSS
Colony PCR AFSS-Topo
Mini prep colonies
Digest plasmid with Aar1
Run diagnostic gel
Mini prep Adh1p colonies
Mini prep Cyc1p colonies
Digest vectors with Aar1
PCR purify
Digest vectors with XcmI
Sticky end PCR (PstI) Lex A- treat with PNK
Run gel; gel purify
Boil; ligate with pRS304-T273M; transform
Colony PCR
Run diagnostic gel; pick up viable colonies
Redo sticky ended PCR for Lex A-PRS304-T273M
Run gel; gel purify
Digest pCAT1 with EcoRV
Transform pCAT1 into CB008 yeast strain
Transform pCAT2 into CB008 strain
Transform pCAT1 and pJAC7 into CB008 strain
Transform pCAT2 and pJAC7 into Lex A-Sir 2 yeast strain
Transform pCAT2 into lex A-Sir 2 strain
Transform pCAT2 and pJAC6 into Lex A-Sir 2 strain
Dilute cultures; take ODs
Design primers for Kpn1 construct
WEEK 6 & 7: 7/21/08-8/3/08
Dilute yeast cultures
Microscopy assay
PCR (KpnI) Lex A
Run gel; gel purify
Digest Lex A-pRS304-T273M with KpnI
Colony PCR Cyc1p colonies
Colony PCR Adh1p colonies
Run diagnostic gel
Digest vectors with XhoI
Run diagnostic gel
Digest; run gel; gel purify
PCR Fus, Fig, Prm
Run gel; gel purify
WEEK 8: 8/4/08-8/10/08
Double digest peptides with NotI and XhoI
Double digest Adh1p-AFSS with NotI and XhoI
Double digest Cyc1p-AFSS with NotI and XhoI
Run gel; gel purify
Ligation; transformation
Mini prep Cyc1p-AFSS-Cecropin A
Mini prep Cyc1p-AFSS-LL37
Mini prep Cyc1p-AFSS-Sarcotoxin A
PCR; digest; run diagnostic gel
Transform plasmids into CB008 yeast strain
Double digest Gal10p with PspOMI and XhoI
Double digest Cyc1p-AFSS-Cecropin A with PspOMI and XhoI
Double digest Cyc1p-AFSS-LL37 with PspOMI and XhoI
Double digest Cyc1p-AFSS-Sarcotoxin A with PspOMI and XhoI
Run gel; gel purify
Ligate; transform
Mini prep Adh1p-AFSS vectors
Double digest with XhoI and NotI
Double digest peptides with XhoI and NotI
Run gel; gel purify
Ligate; transform
Double digest Lex A-Sas 2 with PspOMI and XhoI
Double digest Lex A-Esa I with PspOMI and XhoI
Double digest Lex A-VP64 with PspOMI and XhoI
Double digest mCherry with PspOMI and XhoI
Double digest Prm1p with PspOMI and XhoI
Run gel; gel purify
Ligation; transformation
WEEK 9: 8/11/08-8/16/08
Dilute CB008 Gal10p-AFSS-peptide strains; measure ODs
Colony PCR
Run diagnostic gel
Mini prep plasmids
TCA precipitate samples
Run Western- unsuccessful
WEEK 10 and so on: 8/18/08...
T273M invasive growth assay
Microscopy assay
Restreak and pick up Gal10p strain
Colony PCR
Run diagnostic gel
Subclone Gal10p into 2 micron plasmids
Prepare cultures for bead beating
Western- unsuccessful
Double digest 2 micron vector with PspOMI and SacI
Double digest peptides with PspOMI and SacI
Run diagnostic gel
Ligate; transform
Prepare iGEM presentation
