Team:Stockholm/9 September 2010




Cloning of N-CPPs into pSB1C3

Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.

Digestion of N-CPP cluster

[N-CPP plasmid] = 672 ng/μl

Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.

1st incubation
10X FastDigest buffer 3
DNA (2 μg) 3
dH2O 19
XbaI (conv.) 1
AgeI (conv.) 1
  27 μl
2nd incubation
FD BamHI 1
FD HindIII 1
  29 μl
  • 1st incubation: 37 °C, 2:30
  • 2nd incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 20 min


Two ligation reactions were prepared to test the efficiency of two different ligation buffers.

  • Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
  • Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)
  Lig pSB1C3
Lig pSB1C3
5X Rapid Ligation buf. 4 0
10X T4 DNA ligase buf. 0 2
Vector DNA 4 4
Insert DNA 11 11
dH2O 0 2
T4 DNA ligase 1 1
  20 μl 20 μl
  • Incubation: 22 °C, 16 min

Digestion of previous ligation sample

Ligation mix: Lig pSB1C3.N-CPP* 6/9
Ligation mix 15
10X FD buffer 2
FD BamHI 1
FD HindIII 1
  19 μl
  • Incubation: 37 °C, 30 min
  • Inactivation: 80 °C, 15 min


Standard transformation protocol.

  • 3 μl ligation mix
    • Lig pSB1C3.N-CPP 1 9/9
    • Lig pSB1C3.N-CPP 2 9/9
    • Lig pSB1C3.N-CPP * 6/9
  • Cm 25 plates

Joint expression of SOD and yCCS from pEX

Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by Ahl, Lindberg and Tibell (2004), we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.

Extraction of RBS BioBrick (BBa_B0030)

Extracted BBa_B0030 (RBS 30), carried on pSB1A2, from iGEM plate 1, well 1H. Transformed into Top10.

  • Quick transformation
  • 1 μl DNA
  • Amp 100

Cloning of His⋅SOD into pMA

Sequencing results

  • pMA.his.SOD_premix (fasta)

Correct sequence verified by Blastn (results). Three silent mutations in the His-tag, as has been previously observed.


SOD.his / his.SOD / yCCS

Plasmid prep.

  • Follow E.Z.N.A protocol
    • Wash 2x with DNA wash buffer
    • Eluate in 50µl sH2O

Glycerol stocks

  • Add 1800µl to 200µl pre-sterelized glycerol


Site-Directed Mutagenesis

DNA conc. ng/µl
mix (µl) primers conditions
sH2O 40 MITF_1F time °C
dNTP 1 MITF_1R 2m 95
F primer 1 30s 95 )
R primer 1 30s 55 > 22 cycles
Pfu buffer 5 7m 68 )
Pfu turbo 1 oo 4
tot 50µl

The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/