Team:UCSF/Cathy Liu Notebook

From 2008.igem.org

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Remake AFSS-Topo
Remake AFSS-Topo
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Colony PCR AFSS-Topo
Colony PCR AFSS-Topo
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Mini prep colonies
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Digest plasmid with Aar1
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Run diagnostic gel
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Mini prep Adh1p colonies
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Mini prep Cyc1p colonies
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Digest vectors with Aar1
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PCR purify
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Digest vectors with XcmI
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Run gel; gel purify
Run gel; gel purify
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Boil; ligate with pRS304-T273M; transform
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 +
Colony PCR
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Run diagnostic gel; pick up viable colonies
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 +
Redo sticky ended PCR for Lex A-PRS304-T273M
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Run gel; gel purify
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Digest pCAT1 with EcoRV
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Transform pCAT1 into CB008 yeast strain
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Transform pCAT2 into CB008 strain
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Transform pCAT1 and pJAC7 into CB008 strain
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Transform pCAT2 and pJAC7 into Lex A-Sir 2 yeast strain
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Transform pCAT2 into lex A-Sir 2 strain
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Transform pCAT2 and pJAC6 into Lex A-Sir 2 strain
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 +
Dilute cultures; take ODs
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Design primers for Kpn1 construct
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WEEK 6: 7/21/08 – 7/27/08
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Dilute yeast cultures
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 +
Microscopy assay
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 +
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PCR (KpnI) Lex A
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 +
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
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Run gel; gel purify
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WEEK 7: 7/28/08 – 8/3/08

Revision as of 02:46, 29 October 2008

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: 7/21/08 – 7/27/08

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 7: 7/28/08 – 8/3/08

Retrieved from "http://2008.igem.org/Team:UCSF/Cathy_Liu_Notebook"