Part:BBa_K1887000

LacZ

The lacZ gene from Lactobacillus acidophilus was introduced in the His locus of L. lactis NZ9000 genome by temperature sensitive vector, which makes the NZ-Blue strains become blue on plate with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).

Sequence and Features

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
  COMPATIBLE WITH RFC[1000]

Design

To get device knocked-in recombinant L. lactis without antibiotic resistant genes and make this process easy and highly efficient, we try to establish a visual selection system based on the insoluble blue compounds generated by the lacZ gene encoded β-galactosidase catalyzed X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) hydrolysis. To build this system, we first chose the His locus as the targeting locus, for the His locus is required for the synthesis of histidine, an essential amino acid for L. lactis growth, thus the recombinant L. lactis strains can only be survived with the supplementation of histidine in the culture but can not survive without histine. The His fragment was introduced into a temperature-sensitive conditional replicative shuttle plasmid and we named this plasmid as pHis. Then we placed the lacZ gene driven by the pNisZ promoter into the His locus and we name this plasmid as pBlue. The next step is to integrate the lacZ gene into the genome of NZ9000 to yield the NZ-Blue recombinant strain. The lacZ gene encoded β-galactosidase can hydrolyze the X-gal from colorless substance to insoluble blue compounds, thus NZ-blue strain forms blue colonies on X-gal plate supplemented with nisin. Finally, the device carrying plasmid based on pHis (pDevice) will be introduced to NZ-Blue to replace the lacZ gene and the desired device knocked-in strains will be white when cultured with X-gal and supplemented with nisin.

Results

The temperature-sensitive conditional replicative plasmid pHis was used to introduce the ‘PnisZ::lacZ::Terminator’ fragments into the NZ9000 chromosome of L. lactis. The constructed plasmids pBlue was transformed into NZ9000 by electroporation. To obtain NZ-Blue via homologous sequences (HisF or HisR), the NZ9000-pBlue strains were grown in M17GS (M17 broth with 0.55 % sucrose and 0.5 % glucose) containing erythromycin and incubated at 38.5 °C overnight to integrate the pBlue DNA sequence into the genome of NZ9000 through single cross over. Then, the cultures were diluted in M17GS medium without erythromycin and incubated at 28 °C to promote the double cross over and excision of the antibiotic resistance genes. The subcultures were diluted and plated on M17GS with X-Gal and nisin. Blue colonies obtained by serial plating were screened for erythromycin susceptibility. Colonies in which lacZ gene integration had occurred will appear as blue on X-gal plate supplemented with nisin and erythromycin susceptible. The erythromycin susceptible and blue colonies selected from thousands of colonies were further subject to PCR verification using the extracted genomic DNA as PCR template. DNA sequencing was also used to confirm that no genetic mutations were introduced during the experiment process. After this difficult and tedious task, we successfully got the lacZ integration strain and we name it NZ-Blue.

References

Simoes-Barbosa, A., Abreu, H., Silva Neto, A., Gruss, A., and Langella, P. (2004). A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression. Appl Microbiol Biotechnol 65, 61-67.