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Andreas

Site-directed mutagenesis of SOD & yCCS

Sequencing results

Received the sequencing results for m-SOD 1, m-SOD 2, m-yCCS 1 and m-yCCS 4 from Eurofins. Ran a nucleotide BLAST (BLASTn) alignment to verify successful site-directed mutagenesis.

• SOD 1: Failed (BLASTn results)
  • Mutation still present
• SOD 2: Success (BLASTn results)
  • Mutation removed
• yCCS 1: Success (BLASTn results)
  • EcoRI site removed
• yCCS 4: Success (BLASTn results)
  • EcoRI site removed

All sequences revealed an insertion in the Freiburg suffix, between PstI and SpeI sites. We were aware of this, and the insertion will be removed by digestion later.
BLASTn results also revealed three mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all three mutations as silent mutations (BLASTp results).

SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.

Assembly of CPP⋅protein⋅His constructs

Brief introduction

Our goal is to assemble our CPPs together with our protein-coding genes and a His tag, in one of two ways:

• CPPs with N-part prefix (N-CPP): CPP⋅gene⋅His-tag
• CPPs with Freiburg prefix (C-CPP): His-tag⋅gene⋅CPP

A His-tag (BBa_K157011) is available from the Registry, carried on the pMA (BBa_K157000) plasmid (pMA.His). This has previously been extracted from one of our iGEM plates, and is available both as plasmid, and as transformed cells.
The CPPs have been requested as synthesized DNA genes, and should arrive soon.
At me and Mimmi's lab, yCCS and SOD are now successfully verified in pSB1C3 and ready for cloning.

Assembly and cloning strategies

Cloning strategy for assembly of N-part prefix CPPs (N-CPP) with protein-coding genes and His tag to form N-CPP⋅gene⋅His-tag, into pSB1C3.

Cloning strategy for assembly of Freiburg prefix CPPs (C-CPP) with protein-coding genes and His tag to form His-tag⋅gene⋅C-CPP, into pSB1C3.

Designed cloning strategies for the assembly of CPP⋅gene⋅His-tag and His-tag⋅gene⋅CPP; see drawings attached. We will use BioBrick 3A assembly, with vectors carrying the RFP cassette as out destination vector. pSB1A3, pSB1C3 and pSB1K3 are already available with RFP, but not yet pEX.

Cloning preparations

• New Km 50 LB agar plates
  • 22 pcs
• ON cultures (colonies picked from plates stored in 4 °C, grown in 5 ml LB + antibiotic (pMA & pEX in Amp 100; pSB1K3 in Km 50), 37 °C, 250 rpm, ON)
  • pMA.His
  • pSB1K3.BBa_J04450 (pSB1K3.RFP)
  • pEX

Transfer of RFP into pEX

Digestions

[pEX] = 161 ng/μl

[pSB1C3.RFP] = 148 ng/μl

Proceeded to ligation without enzyme inactivation or DNA purification

Ligation

[DNA] = 66.6 ng/μl

Transformation

Transformation according to quick transformation protocol. 3 μl ligation mix. Cells plated onto 100 Amp LB agar.

Assembly of SOD/yCCS⋅His into pSB1K3

(Step I of N-CPP cloning strategy)

Digestions performed by Mimmi, see section below.
All DNA concentrations in digestion tubes 66.6 ng/μl.

Ligation

Transformation

Quick transformation according to protocol. 3 μl ligation mix. Cells plated onto Km 50 LB agar plates.

Mimmi

SOD / yCCS

Digestion

• Incubate with just NgoMIV in 37°C for 1h 30m
• Add the other restriction enzymes and incubate in 37°C for 30m
• Inactivate restriction enzymes in 80°C for 5m

=Nina

I worked on obtaining material from the Uppsala iGEM-team and also talked to an Uppsala reseach team in Uppsala University Hospital about artificial skin that they might be able to culture and provide to us for testing the penetration capacity of our future fusion proteins with CPP.