Team:UCSF/Cathy Liu Notebook

TEAM OUTPUT AKA: "TEAM DEATH"
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

WHAT HAS BEEN DONE?

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