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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 1.2.1 Extraction of RBS BioBrick (BBa_B0030) 1.3 Cloning of His⋅SOD into pMA 1.3.1 Sequencing results 2 Mimmi 2.1 SOD.his / his.SOD / yCCS 2.1.1 Plasmid prep. 2.1.2 Glycerol stocks 2.2 MITF-M 2.2.1 Site-Directed Mutagenesis

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.

Extraction of RBS BioBrick (BBa_B0030)

Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.

• Quick transformation
• 1 μl DNA
• Amp 100

Cloning of His⋅SOD into pMA

Sequencing results

• pMA.his.SOD_premix (fasta)

Correct sequence verified by Blastn (results). Three silent mutations in the His-tag, as has been previously observed.

Mimmi

SOD.his / his.SOD / yCCS

Plasmid prep.

• Follow E.Z.N.A protocol
  • Wash 2x with DNA wash buffer
  • Eluate in 50µl sH₂O

Glycerol stocks

• Add 1800µl to 200µl pre-sterelized glycerol

MITF-M

Site-Directed Mutagenesis

DNA conc.	ng/µl
pSB1C3.SOD.his
pSB1C3.his.SOD
pSB1C3.SOD.his

mix	(µl)		primers		conditions
sH2O	40		MITF_1F		time	°C
dNTP	1		MITF_1R		2m	95
F primer	1				30s	95	)
R primer	1				30s	55	> 22 cycles
Pfu buffer	5				7m	68	)
Pfu turbo	1				oo	4
DNA	1
tot	50µl