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From 2008.igem.org

12^th week

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Contents 1 Monday 10/20/2008 1.1 pSB1A3-Receiver-Colicin cloning 1.2 Sender cloning: constitutive promotor-sender 2 Tuesday 10/21/2008 2.1 Sequencing results of ready cloned BioBrick parts 2.2 Controldigestion of parts with EcoRI and PstI 2.3 Control of antibiotics resistance 3 Wednesday 10/22/2008 4 Thursday 10/23/2008 4.1 pSB1A2-Receiver-Colicin cloning 4.2 HisTag cloning of Colicins for purification 4.3 Colicin activity test 5 Friday 10/24/2008 5.1 pSB1A3-Receiver-Colicin cloning 5.2 HisTag cloning of Colicins for purification 5.3 Sender Cloning: pBAD - sender 5.4 Sender Cloning: constitutive promotor - sender 6 Saturday 10/25/2008 6.1 Killer-prey system test and lysis test of killer cells 6.2 Theoretical work for documentation 7 Sunday 10/26/2008

Monday 10/20/2008

pSB1A3-Receiver-Colicin cloning

• Minipreps of colE1/E9/E9lys-Receiver with QiaCube, Qiagen
• Send probes to GATC for sequencing

Sender cloning: constitutive promotor-sender

• Minipreps of J23107-Receiver with QiaCube, Qiagen
• Send probe to GATC for sequencing

Tuesday 10/21/2008

Sequencing results of ready cloned BioBrick parts

	EcoRI-site mutation	PstI-site 1 mutation	PstI-site 2 mutation	PstI-site 3 mutation	Prefix	Suffix	complete sequence
colE1_BB_57-1	+	+	+	+	+	+	+
colE1_BB_57-2	+	+	+	+	+	+	+
colE1_BB_57+2	?	+	+	?	+	?	missing sequence
colE1_BB_67+1	+	+	+	+	+	?	sequencing failure
colE9_BB_(2)	+	XXX	XXX	XXX	+	+	+
colE9lys_BB	XXX	XXX	XXX	XXX	+	+	+
sender_BB	XXX	XXX	XXX	XXX	+	+	+

--> colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB gave positive sequencing results in all criteria and will be sent to the registry

Controldigestion of parts with EcoRI and PstI

• colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB was digested with EcoRI and PstI: 1h 30 min -> 37 °C

 0.5 µl EcoRI (NEB)
 0.5 µl PstI (NEB)
 3.0 µl DNA
 2.0 µl EcoRI Buffer (NEB)
 2.0 µl BSA 10x
12.0 µl H2O
-------
20.0 µl

• Gelresults: The digestion pattern looks like expected. 1% Agarose, 135 V, 30 min

Control of antibiotics resistance

• LB-media containing tetracycline, chloramphenicol, ampicilin or kanamycin was inoculated with the different parts to the theri antibiotics resistance.

Wednesday 10/22/2008

• Antibiotics test: Each part only grew in ampicilin media as expected

Thursday 10/23/2008

pSB1A2-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Friday 10/24/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Sender Cloning: pBAD - sender

Sender Cloning: constitutive promotor - sender

Saturday 10/25/2008

Killer-prey system test and lysis test of killer cells

Theoretical work for documentation

Sunday 10/26/2008

Theoretical work for documentation.

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