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From 2010.igem.org

Contents 1 Andreas 1.1 Assembly of new parts 1.1.1 Gel verification of part digestions 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.4 ON cultures 1.2 N-CPP sequencing 1.2.1 ON cultures 2 Mimmi 2.1 SOD / yCCS 2.1.1 protein gel 2.1.2 glycerol stock / over expression 2.1.3 PCR 2.2 MITF-M 2.2.1 colony PCR 2.2.2 Gel 3 Johan

Andreas

Assembly of new parts

Gel verification of part digestions

Ran gels of digestion samples in parallel with undigested samples to verify successful digestions and insert sizes.

Gel 1

1 % agarose, 110 V

Gel 2

1 % agarose, 110 V

Results
Successful digestion with corresponding bands for all digested samples. N-TAT and N-Tra10 not verified, since gel was run too far, but plasmid linearization should indicate successful digestion.

Most samples show somewhat incomplete digestion. For digestions with FastDigest enzymes, this may be an indication of old/inactive enzymes. Especially PstI should be analyzed for activity.

Colony PCR

Picked new colonies for colony PCR from 14/9 plates:

• pSB1K3.N-TAT⋅SOD⋅His: TAT⋅SH 1-5
• pSB1K3.N-Tra10⋅SOD⋅His: Tra10⋅SH 1-5
• pEX.SOD 1-4

Standard colony PCR settings.

• Elongation: 1:30

Gel verification

Gel 1
1 % agarose, 110 V

Expected bands

• pSB1K3.N-TAT⋅SOD⋅His (TAT): 848 bp
• pSB1C3.SOD⋅His (pC.SH): 815 bp

Gel 2
1 % agarose, 110 V

Expected bands

• pSB1K3.N-Tra10⋅SOD⋅His (Tra10): 878 bp
• pSB1C3.SOD⋅His (pC.SH): 815 bp

Gel 3
1 % agarose, 110 V

Expected bands

• pEX.SOD: 678 bp
• pEX.RFP: 862 bp, 1010 bp, 1124 bp, 1272 bp

Results
pSB1K3.N-TAT⋅SOD⋅His clones 4 and 5 seem slightly larger than the control, pSB1C3.SOD⋅His, indicating correct assembly.
Of the pSB1K3.N-Tra10⋅SOD⋅His samples, clone 5 seems correct, compared to the control (same as above). For the pEX.SOD samples the results are very strange. Two of the clones resulted in way too large bands (≈2500 bp); unclear what these vectors carry. Clone 2 resulted in a very weak band with the correct size. This clone was chosen for ON growth.

ON cultures

Set ON cultures (5 ml LB + 50 Km or 100 Amp; 37 °C, 220 rpm) of the following:

• pSB1K3.N-TAT⋅SOD⋅His 4
• pSB1K3.N-TAT⋅SOD⋅His 5
• pSB1K3.N-Tra10⋅SOD⋅His 5
• pEX.SOD 2

N-CPP sequencing

Sequencing results from 13/9 returned.

• pSB1C3.nCCP 2 (fasta)
• pSB1C3.nCCP 3 (fasta)
• pSB1C3.nCCP 5 (fasta)
• pSB1C3.nCCP 8 (fasta)
• pSB1C3.nCCP 9 (fasta)
• pSB1C3.nCCP 10 (fasta)
• pSB1C3.nCCP 11 (fasta)
• pSB1C3.nCCP 12 (fasta)

Ran multiple nucleotide Blast (Blastn) alignments to identify the three N-CPPs from the sequence:

• pSB1C3.N-TAT: clones 9 & 12 (Blastn)
• pSB1C3.N-Tra10: clone 5 (Blastn)
• pSB1C3.N-LMWP: clones 2, 3 & 11 (Blastn)

ON cultures

Set ON cultures for plasmid prep (5 ml LB + 25 Cm; 37 °C, 220 rpm) and glycerol stocks (3 ml LB + 25 Cm; 30 °C).

• Clone 5: pSB1C3.N-Tra10
• Clone 11: pSB1C3.N-LMWP
• Clone 12: pSB1C3.N-TAT

Mimmi

SOD / yCCS

protein gel

• Thaw the samples and re-heat them in 95°C, 5min

• Load them on a PhastGel

• • 20% 8 wells x2

	SOD	yCCS
length	154aa	249aa
Ip	6.0695	6.6594
charge	-2.0	+0.5
size	15.9kDa	27.3kDa

SOD.his	0h	1h	2h	3h
his.SOD	0h	1h	2h	3h
yCCS	0h	1h	2h	3h

well	sample			well	sample
1	ladder			1	ladder
2	SOD.his 0h			2	his.SOD 2h
3	SOD.his 1h			3	his.SOD 3h
4	SOD.his 2h			4	yCCS 0h
5	SOD.his 3h			5	yCCS 1h
6	his.SOD 0h			6	yCCS 2h
7	his.SOD 1h			7	yCCS 3h
8	ladder			8	ladder

glycerol stock / over expression

yCCS

• Pick 2 new colonies, grow ON

SOD.his/his.SOD

• Pick some cells from the glycerol-stock, grow ON

glycerol stocks
yCCS 1 3ml LBAMP + 2µl culture (from PCR)
yCCS 2 3ml LBAMP + 2µl culture (from PCR)
ON for expression
SOD.his 1 5ml LBAMP + 5µl culture (from PCR)
his.SOD 1 5ml LBAMP + 5µl culture (from PCR)
yCCS 1 5ml LBAMP + 5µl culture (from PCR)
yCCS 2 5ml LBAMP + 5µl culture (from PCR)

PCR

mix	(µl)	x4		Primers		conditions
mastermix	24.5			pEX		time	°C
DNA	0.5					10m	95
tot	25µl					30s	95	)
						30s	55	> 30 cycles
						1m	72	)
						5m	72
						oo	25

MITF-M

colony PCR

mix	(µl)	x8		conditions
mastermix	24.5			time	°C
DNA	0.5			10m	95
tot	25µl			30s	95	)
				30s	55	> 30 cycles
				2m	72	)
				10m	72
				oo	25

Gel

well	sample
1	ladder
2	MITF-M 1
3	MITF-M 2
4	MITF-M 3
5	MITF-M 4
6	MITF-M 5
7	nTAT
8	cTAT
9	nTra10

Johan

PCR

• 15sep pMA AS bFGF NS
• 15sep pMA EN bFGF EA

0,5 µl Pol

0,5 µl dNTP

5 µl 5x buffer

1,5 µl for primer

1,5 µl rev primer

16 µl H2O

3 colonies/plate