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From 2010.igem.org

Contents 1 Andreas 1.1 Cloning of N-CPPs into pSB1C3 1.1.1 Digestion of N-CPP cluster 1.1.2 Ligation 1.1.3 Digestion of previous ligation sample 1.1.4 Transformations 1.2 Joint expression of SOD and yCCS from pEX

Andreas

Cloning of N-CPPs into pSB1C3

Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.

Digestion of N-CPP cluster

[N-CPP plasmid] = 672 ng/μl

Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.

	N-CPP
1st incubation
10X FastDigest buffer	3
DNA (2 μg)	3
dH2O	19
XbaI (conv.)	1
AgeI (conv.)	1
	27 μl
2nd incubation
FD BamHI	1
FD HindIII	1
	29 μl

• 1st incubation: 37 °C, 2:30
• 2nd incubation: 37 °C, 0:30
• Inactivation: 80 °C, 20 min

Ligation

Two ligation reactions were prepared to test the efficiency of two different ligation buffers.

• Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
• Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)

	Lig pSB1C3 NCPP 1 9/9	Lig pSB1C3 NCPP 2 9/9
5X Rapid Ligation buf.	4	0
10X T4 DNA ligase buf.	0	2
Vector DNA	4	4
Insert DNA	11	11
dH2O	0	2
T4 DNA ligase	1	1
	20 μl	20 μl

• Incubation: 22 °C, 16 min

Digestion of previous ligation sample

Ligation mix: Lig pSB1C3.N-CPP* 6/9

Ligation mix	15
10X FD buffer	2
FD BamHI	1
FD HindIII	1
	19 μl

• Incubation: 37 °C, 30 min
• Inactivation: 80 °C, 15 min

Transformations

Standard transformation protocol.

• 3 μl ligation mix
  • Lig pSB1C3.N-CPP 1 9/9
  • Lig pSB1C3.N-CPP 2 9/9
  • Lig pSB1C3.N-CPP * 6/9
• Cm 25 plates

Joint expression of SOD and yCCS from pEX

Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by Ahl, Lindberg and Tibell (2004), we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.