Part:BBa_K2233001
lacZ for monitoring yrbD gene expression in B.subtilis chromosome
lacZ and cat (chloramphenicol acetyl transferase) genes flanked by the DNA fragments, which are homologous to the particular regions of B.subtilis (strain NCIB 3610) chromosomal DNA.
This part is designed to insert to the B.subtilis chromosome immediately downstream of yrbD gene via homologous recombination. Upon recombination, lacZ can be fused to yrbD, allowing the yrbD gene expression to be monitored. In this part, lacZ does not include promoter region. Therefore this part has no function by itself unless it is inserted into B.subtilis chromosome. cat gene can be used as a selection marker.
In our project, this part was used in an attempt to measure the concentration of L-theanine by monitoring yrbD gene expression in B.subtilis NCIB 3610 chromosome.
Beta-galactosidase assay
BBa_K2233001 is a lacZ reporter gene designed to be inserted in B.subtilis NCIB 3610 chromosome. In Beta-galactosidase assay, we used beta-galactosidase hydrolase ortho-nitrophenyl-β-D-galactopyranoside (ONPG),the artificial chromogenic substrate. ONPG is colorless, but it turns yellow (λmax = 420 nm) after hydrolased into orthonitrophenol (ONP). Therefore, enzyme activity (gene expression) can be measured using a spectrophotometer. By using BBa_K2233001, we have successfully demonstrated that yrbD, a chromosomal gene in B.subtilis NCIB 3610 could be strongly induced in the presence of L-theanine. For more information, please visit our team wiki. (http://2017.igem.org/Team:Kobe)
Monitoring of yrbD gene expression by BBa_K2233001
(y-axis: enzyme actiity , x-axis; elapsed time after the addition of nitrogen source)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 5376
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 29
Illegal NgoMIV site found at 544 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 5008
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