Team:Stockholm/19 August 2010

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Andreas

Site-directed mutagenesis of SOD & yCCS

Sequencing results

Received the sequencing results for m-SOD 1, m-SOD 2, m-yCCS 1 and m-yCCS 4 from Eurofins. Ran a nucleotide BLAST (BLASTn) alignment to verify successful site-directed mutagenesis.

SOD 1: Failed (BLASTn results)
- Mutation still present
SOD 2: Success (BLASTn results)
- Mutation removed
yCCS 1: Success (BLASTn results)
- EcoRI site removed
yCCS 4: Success (BLASTn results)
- EcoRI site removed

All sequences revealed an insertion in the Freiburg suffix, between PstI and SpeI sites. We were aware of this, and the insertion will be removed by digestion later.
BLASTn results also revealed three mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all three mutations as silent mutations (BLASTp results).

SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.

Assembly of CPP⋅protein⋅His constructs

Brief introduction

Our goal is to assemble our CPPs together with our protein-coding genes and a His tag, in one of two ways:

CPPs with N-part prefix (N-CPP): CPP⋅gene⋅His-tag
CPPs with Freiburg prefix (C-CPP): His-tag⋅gene⋅CPP

A His-tag (BBa_K157011) is available from the Registry, carried on the pMA (BBa_K157000) plasmid (pMA.His). This has previously been extracted from one of our iGEM plates, and is available both as plasmid, and as transformed cells.
The CPPs have been requested as synthesized DNA genes, and should arrive soon.
At me and Mimmi's lab, yCCS and SOD are now successfully verified in pSB1C3 and ready for cloning.

Assembly and cloning strategies

Designed cloning strategies for the assembly of CPP⋅gene⋅His-tag and His-tag⋅gene⋅CPP; see drawings attached. We will use BioBrick 3A assembly, with vectors carrying the RFP cassette as out destination vector. pSB1A3, pSB1C3 and pSB1K3 are already available with RFP, but not yet pEX.

Cloning preparations

New Km 50 LB agar plates
- 22 pcs
ON cultures (colonies picked from plates stored in 4 °C, grown in 5 ml LB + antibiotic (pMA & pEX in Amp 100; pSB1K3 in Km 50), 37 °C, 250 rpm, ON)
- pMA.His
- pSB1K3.BBa_J04450 (pSB1K3.RFP)
- pEX

Transfer of RFP into pEX

Digestions

[pEX] = 161 ng/μl

[pSB1C3.RFP] = 148 ng/μl

Digestion mix
[μl]	pSB1C3.RFP	pEX	Incubation: 37 °C, 30 min
10X FD buffer	3	3
dH₂O	1.5	2.5
2 μg DNA	13.5	12.5
FD XbaI	1	1
FD PstI	1	1
	30	30

Proceeded to ligation without enzyme inactivation or DNA purification

Ligation

[DNA] = 66.6 ng/μl

Ligation mix
100 ng vector (pEX)	1.5	1/3 ratio	Incubation: 22 °C, 10 min
165 ng insert (RFP)	2.5	1/3 ratio
5X Rapid Lig. buffer	4
dH₂O	11
T4 DNA ligase	1
	20

Transformation

Quick transformation as described in protocols. Plated onto 100 Amp LB agar.

Mimmi

SOD / yCCS

Digestion

pSB1C3.SOD/pSB1C3.yCCS			pMA.His		pSB1K3.RFP

8< EcoRI + AgeI			8< NgoMIV + PstI		8< EcoRI + PstI

Mix	SOD	yCCS		His-tag		RFP
sH₂O	7	6		14		11
DNA	19	20		12		15
10X FD buffer	3	3		3		3
EcoRI	0.5	0.5	NgoMIV	0.5	EcoRI	0.5
AgeI	0.5	0.5	PstI	0.5	PstI	0.5
tot	30µl	30µl	tot	30µl	tot	30µl

Incubate with just NgoMIV in 37°C for 1h 30m
Add the other restriction enzymes and incubate in 37°C for 30m
Inactivate restriction enzymes in 80°C for 5m

@@ Line 53: / Line 53: @@
 !pSB1C3.RFP
 !pEX
-|rowspan="7" valign="middle"|''Incubation: 37 &deg;C, 30 min''
+|rowspan="7"|''Incubation: 37 &deg;C, 30 min''
 |-
 |10X FD buffer