Team:UCSF/Cathy Liu Notebook

From 2008.igem.org

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(New page: Objective: Connect silencing/antisilencing to two distinct biological functions (aka "memory readouts")- 1. Cellular differentiation- filamentous growth 2. "Immune response"- antimicrob...)
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c. Test yeast supernatant effects on bacterial growth
c. Test yeast supernatant effects on bacterial growth
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The Road Towards Success
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WEEK 1: 6/23/08-6/29/08
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[Antisilencing work]
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PCR Lex A NLS
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Ligate into Topo; transform
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Mini prep Topo colonies
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Test digest with EcoRI
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Mini prep FRE (Leu) colonies
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Mini prep FRE (Ura) colonies
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Design primers for Dig1 knockout
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WEEK 2: 6/30/08-7/6/08
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Sequence Lex A NLS-Topo
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TEC1* (T273M) transformation
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Mini prep T273M colonies
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Double digest T273M with PstI and KpnI
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Double digest pRS304 vector with PstI and KpnI
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Run gel; gel purify
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Ligate; transform
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Religate digests; transform
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Redo T273M double digest with PstI and KpnI
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Run gel
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Sequence FRE plasmids
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Double digest FRE plasmids with BamHI and ClaI, SacI, EcoRI, or EcoRV
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Run diagnostic gel
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Redigest FRE plasmids
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PCR Dig1 fragments
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Replica plate Lex A-Sir 2-Dig1KO (Nat)
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Replica plate Lex A-Sir 2-Dig1KO (HygB)
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Replica plate CB008-Dig!KO (Nat)
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Replica plate CB008-Dig1KO (HygB)
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WEEK 3: 7/7/08-7/13/08

Revision as of 00:31, 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 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

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