From 2007.igem.org

Contents 1 PCR to perform 2 Oligo design for Wanner deletion 2.1 DapA deletion 2.2 FtsZ deletion

PCR to perform

Perform the following PCR reactions

• P5 (then perform digestion rxn D18 & D20)
• P6 (then perform digestion reactions D19 & D21)
• P8
• P9

Repeat ligation reactions (same as 25/07/07). Also include digestion products D28 & D32

Oligo design for Wanner deletion

We were for now on not able to perform the deletion of DapA through transduction. So we will try the deletion through the Wanner method. We will also attempt the deletion of FtsZ.

We will use the protocol of the keio collection: [http://ecoli.naist.jp/gb6/Resources/deletion/Del_construct.html]

• 20nt of upstream region of kan gene,

5'-ATTCCGGGGATCCGTCGACC-3' (P1)

• 20nt of kan downstream sequence,

5'-TGTAGGCTGGAGCTGCTTCG-3' (P2)

DapA deletion

• 50nt DapA upstream region

5'-CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATG-3' (P3)

• 50nt DapA downstream region (reverse complement)

5'-AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGC-3' (P4)

• dDapA-F

CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATGATTCCGGGGATCCGTCGACC (P3 + P1)

• dDapA-R

AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)

FtsZ deletion

• 50nt FtsZ upstream region

5'-GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATG-3' (P5)

• 50nt FtsZ downstream region (reverse complement)

5'-GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGC-3' (P6)

• dFtsZ-F

GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATGATTCCGGGGATCCGTCGACC (P5 + P1)

• dFtsZ-R

GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)