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From 2010.igem.org

Contents 1 Andreas 1.1 Sequencing results 1.2 Plasmid prep 1.3 Sorting glycerol stocks 2 Nina 2.1 Agarose gel verificaion 2.2 Colony PCR 2.3 Agarose gel verificaion 2.4 Overnight culture 2.5 Digestion 3 Mimmi 3.1 Over expression 4 Johan 4.1 Cut CPP-vector 4.2 Cut his-bFGF 4.3 Ligation his-bFGF into CPP-vector 4.4 Transformation

Andreas

Sequencing results

Sequencing results from 27/9 samples arrived and were analyzed by nucleotide BLAST.

Construct	Sequencing result	Blastn result	Comment
pEX.N-TAT⋅SOD⋅His 3	pEX.nTAT.SOD.his 3_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.N-TAT⋅SOD⋅His 4	pEX.nTAT.SOD.his 4_premix	Blastn	Not verified. Incorrect construct.
pSB1A2.RBS.yCCS 3	pSB1A2.RBS.yCCS 3_premix	Blastn	Partly verified. Three mutations found: (1) Single base deletion in yCCS sequence, causing a frame shift and an extended open reading frame; (2) Insertion between SpeI and PstI, not affecting expression of cloning; (3) Point mutation in PstI site, disabling PstI restriction. Mutations (1) and (3) are both quite unsure, as the sequencing quality in this area is very low. New sequencing from VR will be sent.
pSB1A2.RBS.yCCS 4	pSB1A2.RBS.yCCS 4_premix	Blastn	Partly verified. Four mutations found: (1) Point mutation changing a K codon to T; (2) Single base deletion in yCCS sequence, causing frame shift; (3) Insertion between SpeI and PstI, not affecting expression of cloning; (4) Point mutation in PstI site, disabling PstI restriction. See comment
pSB1K3.N-TAT⋅SOD⋅His 4	pSB1K3.nTAT.SOD.his4_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-TAT⋅SOD⋅His 5	pSB1K3.nTAT.SOD.his5_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-Tra10⋅SOD⋅His 5	pSB1K3.nTra10.SOD.his5_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-LMWP⋅SOD⋅His 1	pSB1K3.nLMWP.SOD.his1_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1C3.N-LMWP⋅SOD⋅His 1	pSB1C3.nLMWP.SOD.his1_premix	Blastn	Not verified. Incorrect construct.
pSB1C3.N-LMWP⋅SOD⋅His 4	pSB1C3.nLMWP.SOD.his4_premix	Blastn	Not verified. Incorrect construct.
pEX.SOD	pEX.SOD_premix	Blastn	Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.yCCS 5	pEX.yCCS 5_premix	Blastn	Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.SOD⋅His	pEX.SOD.his_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.His⋅SOD	pEX.his.SOD_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.

Plasmid prep

From 28/9 ON cultures

Spun down ON cultures 10 min, 3000 x g. Decanted supernatant and stored pellets in -20 °C ON for later plasmid prep.

Sorting glycerol stocks

Started sorting our glycerol stocks into a spreadsheet for future reference.

Nina

Agarose gel verificaion

I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.

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

Arrangement on gel:

From left to right: 1-5 fusion EA, 1-5 Fusion NS, 1-5 IgG LMWP, 1-5 IgG_TAT_N, 1-5 IgG_Tra10_N, 1-5 IgG_TAT_C, 1-5 Protein A_TAT_C, 1-5 pEX, 1-9 pEX

Colony PCR

I ran a new colony PCR on the samples I did not have a possitive result on from the gel above.

The samples for this screen are:

IgG_LMWP_N, _TAT_N, _Tra10_N, protein A_LMWP_N_pex, _TAT_N_pex & _Tra10_N_pex

I screened 8 colonies/dish.

PCR master mix:

• MgCl2 8ul
• phusion buffer 5X 80ul
• dNTP 8ul
• primerR 24ul
• primerF 24ul
• polymerase 8ul
• H2O 240ul

Agarose gel verificaion

I ran an agarose gel 1 % 100 V on the colony PCR products from yesterday to check if I had any inserts.

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

Arrangement on gel:

Overnight culture

I inoculated Fusion EA colony 1 & 3 and Fusion NS colony 1 & 2 each in 12 ml LB with 24 ul chloramphenicol resistance.

Digestion

I digested IgG protease and protein A in the bank vector C in order to perform a gel clean up of the genes and insert them into a pMa-His AS vector.

Digestion:

• H2O 15 ul
• DNA 2 ul
• Fastdigest buffer 10X 2 ul
• Restriction enzyme NgoMIV 1 ul
• Restriction enzyme SpeI 1 ul (Add after 1.5h incubation in 37 °C)

Mimmi

Over expression

• Load gel
  • • dilute 3h samples 1:4
    • load 8µl in the pre-wells
    • the comb takes up 4µl to load on the gel
    • run PhastGel 20%

well	sample		well	sample
1	ladder		1	ladder
2	SOD 0h		2	SOD.his 0h
3	SOD 3h		3	SOD.his 3h
4	yCCS 0h		4	his.SOD 0h
5	yCCS 3h		5	his.SOD 3h

Johan

Cut CPP-vector

5 µl vector ~1µg

1 µl NgoMIV

1 µl EcoRI

2 µl 10x fastbuffer

12 µl H2O

Then heat-inactivation of enzymes

Cut his-bFGF

5 µl his-bFGF (1,5 µl -> 0,3 µg bFGF)

1 µl AgeI

1 µl EcoRI

2 µl 10x fastbuffer

12 µl H2O

Then heat-inactivation of enzymes

Ligation his-bFGF into CPP-vector

5 µl his-bFGF

0,5 µl cpp-vector

2 µl buffer

1 µl T4 ligase

11,5 µl H2O

Transformation

3 µl of all constructs was transformed into top10 cells