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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 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
