Part:BBa_K2688025

J23108_corrected

J23108 promoter is part of the Chris Anderson’s collection of plasmid. The pSB1C3-BBa_J23108 was obtained by PCR mutagenesis of the pSB1C3-BBa_J23119.

This biobrick is a corrected version of the initial biobrick (BBa_J23108) obtained from (2018 – Plate 4 – 4C) position which contains extra-sequences (notably SpeI forbidden sites).

This corrected version is devoid of extra-sequences and is therefore convenient for a direct biobrick cloning of RBS-CDS under the control of J23108 promoter.

For details on the process, see http://2018.igem.org/Team:GO_Paris-Saclay/Improve

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization of the Anderson promoters cloned within pSB1C3

We took the four pSB1C3 plasmids harbouring an Anderson promoter, introduced them in DH5α bacteria by chemotransformation, miniprepared their DNA and sequenced them. Indeed we observed that the plasmid harboring BBa_J23119 promoter was the only one to present the expected sequence (prefix –BBa_J23119- suffix). The 3 other plasmids contain extra sequences between the promoter sequence and the prefix and suffix sequences that flank the BioBrick (Figure 2).

Figure 1: Multiple sequence alignment of four Anderson promoters of interest

We can notice that two extra SpeI sites are present within the extra sequences surrounding the BBa of interest. This is detrimental for further BioBrick cloning strategy (using SpeI) performed within pSB1C3_BBa_Anderson_promoter (BBa_J23108, BBa_J23109 or BBa_J23111) vectors since SpeI sites are present before and after the promoter sequence within this vector (Figure 2). Improvement of pSB1C3-BBa_J23108

We notice that only one bp was different between BBa_J23108 and BBa_J23119 (Figure 1). Therefore we took advantage of the fact that pSB1C3- BBa_J23119 was correct (without extra sequences between the promoter and the prefix and suffix flanking regions) to obtain by directed mutagenesis a pSB1C3- BBa_ J23108 without extra sequences. The pSB1C3- BBa_J23119 was amplified by PCR using GO2018-17 (CTGACAGCTAGCTCAGTCCTAG) and GO2018-18 (CTCTAGAAGCGGCCGCGAATTC) primers. The PCR product was digested by DpnI (to eliminate the parental pSB1C3- BBa_J23119 plasmid), phosphorylated and auto-ligated. After chemotransformation of DH5α bacteria and minipreparation of plasmidic DNA of selected clones, we checked the genetic organization of the resulting plasmid by digestion and sequencing (Figure 3).

Conclusion: We obtained the pSB1C3-J23108_corrected (BBa_K2688025) Biobrick without extra sequences that is suitable to directly perform cloning under the control of BBa_ J23108 using a BioBrick assembly (with SpeI-PstI). This will be helpful to enlarge the use the Anderson promoter collection for direct cloning within pSB1C3.