IGEM:Paris Bettencourt 2012/Notebooks/RE group/Construction design

Fse gene (gBlocks Gene Fragments)

1. Gene sequence
   • Full: [Frankia sp. Eul1b FseI restriction-modification system gene cluster, complete sequence]
   • Without methyltransferase: [Frankia sp. Eul1b FseI restriction-modification system gene cluster, complete sequence]
2. Check usage in E.Coli
3. Check the lack of:
   • EcoRI [E]
   • Xbal [X]
   • SpeI [S]
   • PstI [P]
   • FseI
4. Codon optomozation
5. ATG...TAA
6. Salis Lab Calculator (not the strongest RBS)

We are planing FokI restriction site to devide sequence (almost in the middle).

RBS_FseI gBlocks construct

Complete Construct

Media:RBS_FseI.geneious

Fragments 1

Media:RBS_FseI_frg1.geneious

Fragements 2

Media:RBS_FseI_frg2.geneious

RBS_IsceI gBlocks construct

• Complete Construct: Geneious file : Media:RBS_IsceI.geneious
• Fragments 1: Media:RBS_IsceI_frg1.geneious
• Fragements 2: Media:RBS_IsceI_frg2.geneious

Sequences

Prefix 1

Before sequences starting with ATG GAATTCGCGGCCGCTTCTAG

Prefix 2

Before sequences starting with no ATG GAATTCGCGGCCGCTTCTAGAG

Suffix

TACTAGTAGCGGCCGCTGCAG

Plasmid 1 : pSB3C5_Pbad_RBS_FseI

• pbad : BBa_I13453
• RBS : BBa_B0032
• FseI: [1]

We used gBlocks to synthetize RBS_FseI

Geneious file : Media:Plasmid_Pbad_RBS_FseI.geneious (right click)

Plasmid 2 : pSB3C5_Pbad_RBS_IsceI

• pbad : BBa_I13453
• RBS : BBa_B0032
• IsceI:

We used gBlocks to synthetize RBS_IsceI

Geneious file : Media:Plasmid_Pbad_RBS_IsceI.geneious (right click)

Plasmid 3 : pSB3C5_Plac_RBS_FseI

• Plac : BBa_R0011
• RBS : BBa_B0032
• FseI: [2]

We used gBlocks to synthetize RBS_FseI

Geneious file : Media:Plasmid_Plac_RBS_FseI.geneious (right click)

Plasmid 4 : pSB3C5_Plac_RBS_IsceI

• plac : [3]
• RBS : BBa_B0032
• IsceI:

We used gBlocks to synthetize RBS_IsceI

Geneious file : Media:Plasmid_Plac_RBS_IsceI.geneious (right click)