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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 2 Nina 2.1 Mini prep 2.2 Protein induction 2.3 Making solution 2.4 Verification of ligation 3 Mimmi (At Biochemistry) 3.1 Over expression

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

Nina

Mini prep

I performed a mini prep according to the procedure described in protocols.

The experiment was carried out on colony # 1 from protein A _LMWP_N, _TAT_N, _Tra10_N, Fusion _CPP1, _TAT_C, _CPP3, Fusion_LMWP_N, _TAT_N & _Tra10_N

Protein induction

I prepared a culture of SOD.His (both N & C terminal), SOD_TAT_N, SOD_LMWP_N & SOD_Tra_10 to reach OD600 = 0.6. Then I induced these with IPTG 10 ul/sample.

I took samples at 0, 1, 2 & 3 h, centrifuged 1 min 13000 rpm, resuspended the pellet with 50 ul water and added 50 ul RDSB (with DTT) and stored in a freezer until suitable for loading into an SDS-gel.

Making solution

I made some solutions of RDSB to have in the freezer when loading protein induction on gel.

Werners sample buffer:

• SDS 4.5 g
• Glycerol 15 % 7.5 ml
• Tris pH 7.8 (30 mM) 1.5 ml (1M)
• Bromophenol Blue (a tip of a spoon)
• H2O up to 50 ml

900 ul Werners sample buffer + 100 ul DTT (1M)

Verification of ligation

I ran an agarose gel 1 % 100 V to check if the ligation of IgG_Tra10_N in the pEX vector was successful, but when I looked at the gel there were no bands at all. I must have missed to add the vector and the gene in the ligation.

Mimmi (At Biochemistry)

Over expression

• Samples
  • 1ml culture
  • centrifuge at 13,000rpm for 1min
  • remove supernatant
  • resuspend pellet in 50µl H₂O
  • add 50µl sample buffer
  • sonicate sample (? for 30s)
  • heat sample at 95°C for 5min
  • centrifuge sample and load supernatant

• Western blot/Comassie 3 gels
• SDS-Page 15%
  • Acrylamide 15ml
  • Resolving buffer 7.5ml
  • H₂O 7.5ml
  • SDS 20% 150µl
  • TEMED 15µl
  • 10% amps 90µl

• Stacker
  • Acrylamide 670µl
  • Stacking buffer 1250µl
  • H₂O 3ml
  • SDS 20% 25µl
  • TEMED 10µl
  • 10% amps 37µl