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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX 1.1.1 Digestions 1.1.2 Ligations 1.1.3 Transformation 1.2 Transformation of BL21 1.3 ON cultures 2 Nina 2.1 Send for sequencing 2.2 Digestion of protein A and peX vector 2.3 Agarose gel on digests 2.4 Miniprep 2.5 Overnight culture

Andreas

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

Digestions

1. pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
   • Clones 2 & 3
2. pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
   • Clones 1 & 2
3. pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
   • Clones 2 & 3

	1:2	1:3	2:1	2:2	3:2	3:3
10X FastDigest buffer	2	2	2	2	2	2
DNA (1 μg)	5.2	4.1	2.5	3.3	4	4.1
dH2O	10.8	11.9	13.5	12.7	12	11.9
FD XbaI	1	1	1	1	1	1
FD PstI	1	1	1	1	1	1
	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl

• Incubation: 37 °C, 1.45
• Inactivation: 80 °C, 20 min

Ligations

• Vector: [Dig pEX.RFP X+P

	pEX.1:2	pEX.1:3	pEX.2:1	pEX.2:2	pEX.3:2	pEX.3:3
10X T4 Ligase buffer	2	2	2	2	2	2
Vector DNA	1.5	1.5	1.5	1.5	1.5	1.5
Insert DNA	8	8	8	8	8	8
dH2O	7.5	7.5	7.5	7.5	7.5	7.5
T4 DNA ligase	1	1	1	1	1	1
	20 μl	20 μl	20 μl	20 μl	20 μl	20 μl

• Incubation: 22 °C, 15 min

Transformation

Quick transformation

• 1 μl ligation mix
• 50 μl 0.1 M IPTG
  • pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
  • pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
  • pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
  • pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
  • pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
  • pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)

Transformation of BL21

Quick transformation

• 50 μl competent cells
• 0.5 μl plasmid
  • pEX.nTra10⋅SOD⋅His
  • pEX.nLMWP⋅SOD⋅His

ON cultures

• 3 ml LB + appropriate antibiotic; 30 °C
  • pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
  • pSB1K3.BBa_J04450 (Top10; Km 50)

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Nina

Send for sequencing

I sent samples for sequencing and the mixtures were 15 ul sample and 1.5 ul forward bank vector verification primer.

• Protein A in LMWP_Ntermin ASB0045 680
• Protein A in TAT_Ntermin ASB0045 679
• Protein A in Tra10_Ntermin ASB0045 678

Digestion of protein A and peX vector

I got a mini prep of the peX vector from Andreas with a concentration of 55.52 ng/ul. I cut this vector and protein A in CPPs_N vectors.

peX:

• Fast digest buffer 10X 3.4 ul
• DNA 30 ul
• Restriction enzyme XbaI 2 ul
• Restriction enzyme PstI 2 ul

Incubated in 37 °C for 30 min.

Protein A:

• Fast digest buffer 10X 2.2 ul
• DNA 20 ul
• Restriction enzyme XbaI 1 ul
• Restriction enzyme PstI 1 ul

Incubated in 37 °C for 30 min.

Agarose gel on digests

I ran the digested products on an agarose gel 1 % 100 V.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Arrangement on gel:

Miniprep

I performed a mini prep on Fusion EA # 1 and 3. Fusion AS I have to put a new overnight culture of since I accidentally dropped the solution and the material is now lost.

The procedure was according to the method described in protocols.

Overnight culture

I ioculated IgG protease_Tra10 # 4 & 6 in each 12 ml LB falcon tubes with 24 ul chloramphenicol.