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From 2010.igem.org

Contents 1 Morning meeting 2 Andreas 2.1 Expression of SOD and yCCS from pEX expression vector 2.2 Preparation of competent BL21(DE3) cells 3 Mimmi 3.1 CPP's 3.1.1 designing primers for assembly standard 25 3.1.1.1 Transportan10 3.1.1.2 LMWP 3.1.1.3 TAT (dowdy's) 4 Johan

Morning meeting

We discussed the project proceedings.

• It was decided that already amplified genes (cloned in pEX vector) should be transferred to [http://partsregistry.org/Part:pSB1C3 pSB1C3] vector.
  • IgG protease
  • Superoxidase dismutase (SOD)
    • yCCS
  • bFGF
• Primers for not-yet amplified genes should be redesigned to include the complete Assembly standard 25 prefix and suffix.
  • CPP
    • Transportan 10
    • LMWP
    • TAT
  • MITF
  • Protein A Z-domain
  • Tyrosinase
  • Vitamin B9 genes
• BL21(DE3) was chosen as our strain for IPTG-induced protein expression from pEX vector.

Andreas

Expression of SOD and yCCS from pEX expression vector

Continued from 29/6

We realized that Top10 and DH5alpha are not suitable for IPTG-induced protein expression. SOD and yCCS expression was therefore not proceeded from ON cultures. Glycerol stocks were prepared from ON culture and pEX.SOD and pEX.yCCS plasmids were prepared.

DNA concentrations

Sample	DNA conc (ng/ul)	A(260)/A(280)
pEX.SOD	18.15	1.98
pEX.yCCS	20.55	1.97

Preparation of competent BL21(DE3) cells

• 5 ml LB was inoculated with BL21(DE3) cells and grown ON in 37°C, 250 rpm.

Mimmi

CPP's

designing primers for assembly standard 25

• No start or stop codon

• From the pSB1AsF:

• Only the length can be ajusted, too short with 16 bases?

Transportan10

• Probably good to bind in the C-terminal end (not tested):

To sit in the N-terminal end of the protein of interest:

• Sequence the right way, use the n-part prefix

G . . AATTC--------------114---------------CTGCA . . G

CTTAA . . G--------------106---------------G . . ACGTC

To sit in the C-terminal end of the protein of interest:

• turn the aa sequence around -> the DNA sequence around, use the normal prefix

LMWP

• Binding in the N-terminal (as done in the study):

To sit in the N-terminal end of the protein of interest:

• turn the aa sequence around -> the DNA sequence around, use the n-part prefix

To sit in the C-terminal end of the protein of interest:

• Sequence the right way, use the normal prefix

TAT (dowdy's)

• Binding in both terminals (neither is done in the study):

Johan

• Miniprep

Promega plasmid yield

2 tubes bFGF, 3 ml from each (maximum for that protocol)

Abs: 20 ng/µl & 70 ng/µl

• Miniprep

- Rest of bFGF (12 ml in total) - 12 ml * 5 pEX