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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 2 Nina 2.1 Mini prep on IgG_Tra10_N#6 2.2 Overday culture of SOD 2.3 Gene digestions 2.4 Agarose gel on digests 2.5 Gel clean up 2.6 Concentration measurments 2.7 Sending for sequencing 3 Mimmi 3.1 Protein purification

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
  • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: 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

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Nina

Mini prep on IgG_Tra10_N#6

I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.

Overday culture of SOD

I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.

The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.

Gene digestions

I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.

Digestions:

• Fusion_EA_His # 1 & 3 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• Fusion_NS_His # 2 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme AgeI 1 ul
• Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)

• Protein A_EA_His # 5 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• IgG protease_EA_His # 5 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• IgG protease_Tra10_Ntermin # 4 & 6 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme XbaI 1 ul
• Restriction enzyme PstI 1 ul

Incubate in 37 °C for 30 minutes.

Agarose gel on digests

I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.

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

Arrangement on gels:

Gel clean up

I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel.

I performed the clean up according to the method described in protocols.

The measurements of the cut gel bands, addition of kit solutions and incubation time:

• All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI

• I added 10 ul of QIAEXII to all samples

• All samples were incubated for 5 minutes at RT

• I performed step 11 in the procedure description

Concentration measurments

I measured with a spectrophotometer the concentration of the gel clean up samples.

Sending for sequencing

I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).

• ASB0045 898

Mimmi

Protein purification

• Using Qiagen Ni-NTA Spin Kit

Buffers

Lysis buffer NPI-10 Wash buffer NPI-20	NaH2PO4•H2O NaCl + imidazole	3.45g 8.77g 6.8g	50mM 300mM 2M	\ / 500ml 100ml
Elution buffer	NaH2PO4•H2O NaCl imidazole	3.45g 8.77g 17g	50mM 300mM 500mM	) > 500ml )
Lysosyme		0.1g	10mg/ml	10ml