Part:BBa_K1152014:Design

indC-ccdB

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4572
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1467
  Illegal BamHI site found at 3742
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3192
  Illegal SapI.rc site found at 2730
  

Design Notes

The native indC contains an internal EcoRI and SpeI cutting site which was mutated using a CPEC approach. We amplified indC in three fragments from P. luminescens genomic DNA:

1. start codon to EcoRI cutting site (Primer 1/2; Table 1)
2. EcoRI cutting site to SpeI cutting site (Primer 3/4; Table 1)
3. Spe cutting site to stop codon (Primer 5/6; Table 1).
   

The primers contain point mutation (indicated with small letters in Table 1) to remove the cutting sites. The backbone was amplified with the primers 7/8. Basically every backbone which contains the part J04450 can be used.

Table 1: Primer for removal of internal RFC[10] cutting sites in indC using a CPEC approach

Name	Primer Sequence 5' - 3'
1_indC_fw	CATACTAGAG AAAGAGGAGAAA GGTACC ATGTTAGAAAATAATATTACACAATG
2_EcoRI_rv	CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC
3_EcoRI_fw	AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC
4_SpeI_rv	ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG
5_SpeI_fw	CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC
6_indC_rv	CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG
7_backbone_fw	TAATGA GCTAGC TAATAACGCTGATAGTGCTAGTG
8_backbone_rv	CAT GGTACC TTTCTCCTCTTT CTCTAGTATGTGTG
9_K1152013_fw	AAGTGGATTGAACAGACAGACTCTAAAAC
10_K1152013_rv	AGTATCTGTATGTAATGGCACCAATAGACGC
11_ccdB_fw	TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC
12_ccdB_rv	AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC

In a 6 ul PCR Master Mix (NEB Phusion High-Fidelity in High Fidelity buffer), all fragments were put together in an equimolar ratio. CPEC assembly was performed using an optimized protocol (Table 2).

Table 2: CPEC assembly Cycler Parameters

Cycles	Temperature [°C]	Time [s]
1	98	30
5	98	5
	53	15
	72	60
1	72	180

Transformation was performed with 5 ul of the CPEC reaction product. The resulting plasmid is K1152008. We subsequently amplified the plamid without the T-domain with primers 9/10 and the ccdB cassette from pDONR with primers 11/12. In a second CPEC assembly, the indC-ccdB part was created and transformed into OneShot ccdB survival cells. The prepped plasmid was then transformed into both E. coli TOP10 and OneShot cells, outlining that there are no background colonies (Figure 1).

Using the same strategy as for introducing ccdB, we introduced the plu2642 T-domain with the respective primers.

Source

The coding sequence was amplified from Photorhabdus luminescens laumondii TT01 DSM15139.

References

1. Brachmann AO, Kirchner F, Kegler C, Kinski SC, Schmitt I, Bode HB (2012) Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens. J Biotechnol 157: 96-99.
2. Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4: e6441.