Team:UCSF/Cathy Liu Notebook

From 2008.igem.org

(Difference between revisions)
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Mini prep Topo colonies
Mini prep Topo colonies
Test digest with EcoRI
Test digest with EcoRI
 +
Mini prep FRE (Leu) colonies
Mini prep FRE (Leu) colonies
Mini prep FRE (Ura) colonies
Mini prep FRE (Ura) colonies
 +
Design primers for Dig1 knockout
Design primers for Dig1 knockout
 +
Line 55: Line 58:
TEC1* (T273M) transformation
TEC1* (T273M) transformation
 +
Mini prep T273M colonies
Mini prep T273M colonies
 +
Double digest T273M with PstI and KpnI
Double digest T273M with PstI and KpnI
 +
Double digest pRS304 vector with PstI and KpnI
Double digest pRS304 vector with PstI and KpnI
 +
Run gel; gel purify
Run gel; gel purify
 +
Ligate; transform
Ligate; transform
 +
Religate digests; transform
Religate digests; transform
-
Redo T273M double digest with PstI and KpnI
+
 
 +
Redo pRS304-T273M double digest with PstI and KpnI
 +
 
Run gel
Run gel
 +
Sequence FRE plasmids
Sequence FRE plasmids
 +
Double digest FRE plasmids with BamHI and ClaI, SacI, EcoRI, or EcoRV
Double digest FRE plasmids with BamHI and ClaI, SacI, EcoRI, or EcoRV
 +
Run diagnostic gel
Run diagnostic gel
 +
Redigest FRE plasmids
Redigest FRE plasmids
PCR Dig1 fragments
PCR Dig1 fragments
 +
 +
Replica plate Lex A-Sir 2-Dig1KO (Nat)
Replica plate Lex A-Sir 2-Dig1KO (Nat)
 +
Replica plate Lex A-Sir 2-Dig1KO (HygB)
Replica plate Lex A-Sir 2-Dig1KO (HygB)
 +
Replica plate CB008-Dig!KO (Nat)
Replica plate CB008-Dig!KO (Nat)
 +
Replica plate CB008-Dig1KO (HygB)
Replica plate CB008-Dig1KO (HygB)
 +
WEEK 3: 7/7/08-7/13/08
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
 +
 +
 +
 +
WEEK 4: 7/14/08-7/20/08

Revision as of 00:50, 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


WEEK 4: 7/14/08-7/20/08

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