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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of nCPP⋅SOD⋅His.RBS.yCCS operons to pEX 1.1.1 Sequencing 1.2 Removal of insertion in BioBrick suffixes 1.2.1 Digestions 1.2.2 Gel verification 1.2.3 Gel extraction 1.2.4 ON culture 1.3 Colony PCR of nCPP⋅SOD⋅His.RBS.yCCS operons in BL21 1.3.1 Gel verification 1.3.2 ON cultures 1.4 Growth curve assay 1.4.1 ON cultures

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operons to pEX

Sequencing

15 μl plasmid DNA, 1.5 μl primer

• pEX.nTra10⋅SH.Ry_pEXf: ASB0045 768
• pEX.nTra10⋅SH.Ry_pEXr: ASB0045 769
• pEX.nTAT⋅SH.Ry_pEXf: ASB0045 770
• pEX.nTAT⋅SH.Ry_pEXf: ASB0045 771
• pEX.nLMWP⋅SH.Ry_pEXf: ASB0045 772
• pEX.nLMWP⋅SH.Ry_pEXf: ASB0045 773

• 15 μl plasmid DNA; 1.5 μl primer

DNA concentration
Sample	Primer	Label	Sequence code
pEX.nTra10⋅SOD⋅His.RBS.yCCS	pEXf	pEX.nTra10⋅SH.Ry_pEXf	ASB0045 768
pEX.nTra10⋅SOD⋅His.RBS.yCCS	pEXr	pEX.nTra10⋅SH.Ry_pEXr	ASB0045 769
pEX.nTAT⋅SOD⋅His.RBS.yCCS	pEXf	pEX.nTAT⋅SH.Ry_pEXf	ASB0045 770
pEX.nTAT⋅SOD⋅His.RBS.yCCS	pEXr	pEX.nTAT⋅SH.Ry_pEXr	ASB0045 771
pEX.nLMWP⋅SOD⋅His.RBS.yCCS	pEXf	pEX.nLMWP⋅SH.Ry_pEXf	ASB0045 772
pEX.nLMWP⋅SOD⋅His.RBS.yCCS	pEXr	pEX.nLMWP⋅SH.Ry_pEXr	ASB0045 773

Removal of insertion in BioBrick suffixes

An insertion between SpeI and PstI present in IgGp, bFGF, ProtA, yCCS and SOD needs to be removed before submission to the Registry. This will be done by digestion with SpeI (inside insertion) and moving digested gene into a new vector.

Digestions

No sample of SOD available for the moment, so this will be digested after plasmid prep.

	pC.IgGp (A)	pC.bFGF (B)	pC.ProtA (C)	pC.yCCS (D)	pC.RFP (E)
10X FastDigest buffer	2	2	2	2	2
DNA	6	10	16	14	11
dH2O	10	5	0	2	5
FD SpeI	1	1	1	1	1
FD EcoRI	1	1	1	1	1
	20 μl	20 μl	20 μl	20 μl	20 μl

• Incubation: 37 °C, 30 min

Gel verification

1 % agarose, 130 V

IgGp bFGF ProtA yCCS RFP

Expected bands

• pSB1C3.IgGp (A)
  • Vector: 2050 bp
  • Insert: 990 bp
• pSB1C3.bFGF (B)
  • Vector: 2050 bp
  • Insert: 520 bp
• pSB1C3.ProtA (C)
  • Vector: 2050 bp
  • Insert: 230 bp
• pSB1C3.yCCS (D)
  • Vector: 2050 bp
  • Insert: 800 bp
• pSB1C3.RFP (E)
  • Vector: 2050 bp
  • Insert: 1120 bp

Results
Relevant-sized for all samples showing successful (but incomplete) digestion. Proceeded to gel extraction.

Gel extraction

Loaded remaining 17 μl of each sample on a new 1 % agarose gel. Relevant bands excised by gel extraction and saved in -20 ° for later purification.

ON culture

Set ON culture of pSB1C3.SOD for later plasmid prep.

• 5 ml LB + Cm 25; 37 °C, 225 rpm

Colony PCR of nCPP⋅SOD⋅His.RBS.yCCS operons in BL21

1. BL21 pEX.nTra10⋅SOD⋅His.RBS.yCCS (A & B)
2. BL21 pEX.nTAT⋅SOD⋅His.RBS.yCCS (A & B)
3. BL21 pEX.nLMWP⋅SOD⋅His.RBS.yCCS (A & B)

• Standard colony PCR settings.
• Elongation: 1:30 (too short?)

Gel verification

1 % agarose, 130 V

Expected bands

1. 1553 bp
2. 1523 bp
3. 1532 bp

Results
Bands with relevant sizes for all clones.

ON cultures

3 ml LB + Amp 100

1. B
2. B
3. B

Growth curve assay

A growth curve assay will be run to test the toxicity of our CPPs.

ON cultures

5 ml LB + Amp 100; 37 °C, 225 rpm

• BL21, pEX.SOD⋅His
• BL21, pEX.nTra10⋅SOD⋅His
• BL21, pEX.nTAT⋅SOD⋅His
• BL21, pEX.nLMWP⋅SOD⋅His