Part:BBa_K1804001:Design

GFP under the control of constitutive promoter lacI

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 734
  

Design Notes

plac-gfp was constructed by prepending the KpnI sequence and appending the XhoI sequence to the start and stop codons of gfp (BBa_E0040 [1]) respectively. This was done via extension polymerase chain reaction (PCR). Restriction enzyme digest with KpnI and XhoI was then carried out to replace esaR and esaI with the PCR product in the pAC-EsaR-EsaI plasmid (Figure 1). This placed gfp under the control of the plac promoter. The resultant pAC-plac-gfp plasmid encoded gfp under the control of plac.

Figure 1: A. Plasmid map of pAC-EsaR-EsaI plasmid (Addgene plasmid #47660) with restriction sites. B. Plasmid map of pAC-plac-gfp.

The BioBricks Prefix and Suffix were then prepended and appended to the start of plac and end of gfp specific primers, respectively, to produce BBPrefix-plac-gfp-BBSuffix by PCR. To increase the percentage of cleavage during restriction digest, primers with short (referred to as junk) sequences [2] prepended to BBPrefix (FP_BBP_Junk) and appended to BBSuffix (RP_BBS_Junk) were used to amplify BBPrefix-plac-gfp-BBSuffix by PCR. The resultant PCR product Junk-BBPrefix-plac-gfp-BBSuffix-Junk was then cloned into pSB1C3 by restriction digest with EcoRI and PstI followed by ligation and transformation into E. coli BL21 to produce plac-gfp in pSB1C3 (Figure 2). Colony PCR was performed with the BBPrefix and BBSuffix primers to confirm if clone was correct. Figure 3 shows the gel electrophoresis of the PCR products formed after PCR of the correct pSB1C3-plac-gfp clone, PCR-purified Junk-BBPrefix-plac-gfp-BBSuffix-Junk (positive control), pAC-plac-gfp (negative control) and water (negative control) using the BBPrefix and BBSuffix primers.

Figure 2: Plasmid map of plac-gfp in pSB1C3 vector.

Figure 3: PCR of a correct pSB1C3-plac-gfp clone (lane 1), PCR-purified Junk-BBPrefix-plac-gfp-BBSuffix-Junk (positive control) (lane 2), pAC-plac-gfp (negative control) (lane 3) and water (negative control) (lane 4) using the BBPrefix and BBSuffix primers. Marker used was 100 bp ladder.

Source

The lacI promoter (plac) was obtained from the pAC-EsaR-EsaI plasmid (Shong & Collins, 2013), which was a gift from Cynthia Collins (Addgene plasmid # 47660).

The Green Fluorescence Protein (GFP) gene was obtained from BioBricks Part BBa_E0040 [3].

References

Shong, J., & Collins, C. H. (2013). Engineering the esaR promoter for tunable quorum sensing-dependent gene expression. ACS Synthetic Biology, 2(10), 568-575.