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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of pEX.nCPP⋅SOD⋅His to BL21 1.1.1 Gel verification 1.1.2 ON cultures 1.2 Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX 1.2.1 Colony PCR 1.2.2 Gel verification 1.2.3 ON cultures 1.3 Verification of pSB1x3 plasmids 1.4 Assembly of His⋅SOD⋅cCPP constructs 1.4.1 Ligations 1.4.2 Transformations

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Re-run of 2/10 BL21 samples

1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
2. BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

1. 744 bp
2. 765 bp

Results

1. Both clones verified
2. Clone A verified; weak band for clone B

ON cultures

• 3 ml LB, 30 °C
  1. A: BL21 pEX.nLMWP⋅SOD⋅His
  2. A: BL21 pEX.nTra10⋅SOD⋅His

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

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

• 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
• 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

• Elongation time: 2:00

Gel verification

0.8 % agarose, 100 V

Expected bands:

• 5. 1553 bp
• 6. 1553 bp

Results

• 5. Relevant band for clone B; too large insert (double?) for clone A.
• 6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

• 5 ml LB, 37 °C, 250 rpm
  • pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
  • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

• pSB1A3
• pSB1C3
• pSB1K3
• pSB1AC3
• pSB1AK3

Assembly of His⋅SOD⋅cCPP constructs

Continued from 1/10

Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.

Ligations

• Vectors:
  1. Dig pSB1C3.cTra10 E+N
  2. Dig pSB1C3.cTAT E+N
  3. Dig pSB1C3.cLMWP E+N
• Insert: Dig pMA.His⋅SOD E+A

	1	2	3
10X T4 Ligase buffer	2	2	2
Vector DNA	1	1	1
Insert DNA	5	5	5
dH2O	11	11	11
T4 DNA ligase	1	1	1
	20 μl	20 μl	20 μl

• Incubation: 22 °C, 15 min

Transformations

1. pSB1C3.His⋅SOD⋅cTra10
2. pSB1C3.His⋅SOD⋅cTAT
3. pSB1C3.His⋅SOD⋅cLMWP

• Standard transformation
  • 1 μl
  • Cm 25