Team:Stockholm/9 September 2010

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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 [http://www.ncbi.nlm.nih.gov/pubmed/15358352 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.