ybiJ promoter coupled to mKate2 reporter gene and Rhamnose promoter coupled to N-genes
Assembly of a part from Belkin laboratory (Belkin et al. Appl Microbiol Biotechnol (2014) 98:885???895) with a "de novo" synthesised part
Promoter of the ybiJ gene, which is activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli. nfsA, nfsB and nemA (refered here as N-genes) are the genes for NfsA, NfsB and NEM reductases, which presumably play a role in 2,4-DNT and 2,4,6-TNT metabolism. These genes are regulated by the inducible Rhamnose promoter This construct is, then, designed to be able to detect and quantify the presence of compunds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. The presence of the N-genes aims to enhance this response.
The Rhamnose promoter was used to inducibly regulate the expression of the N-genes A double transcriptional terminator was placed between mKate2 and the N-genes in orther to avoid unfinished transcripts of the N-genes, as well as to avoid expression of these genes without the presence of rhamnose A strong RBS was placed in front of each gene. An "all frame STOP" region was placed after each N-gene. nfsA and nfsB have a 6xHis tag at the N-terminus; nemA has a 6xHis tag at the C-terminus. This way, the proteins can be purified to check if they are successfully expressed
ybiJ promoter coupled to mKate2 reporter gene
Combination of BBa_K1316004 with BBa_K1316001
Promoter of the ybiJ gene, which is activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli. This construct is, then, designed to be able to detect and quantify the presence of compunds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB.
The sequence was positively checked for the absence of illegal restriction sites. It is, therefore, biobrick-compatible
nfsA, nfsB and nemA (refered here as N-genes) are the genes for NfsA, NfsB and NEM reductases, which presumably play a role in 2,4-DNT and 2,4,6-TNT metabolism. These genes are regulated by the inducible Rhamnose promoter
The Rhamnose promoter was used to inducibly regulate the expression of the N-genes A strong RBS was placed in front of each gene An "all frame STOP" region was placed after each gene nfsA and nfsB have a 6xHis tag at the N-terminus; nemA has a 6xHis tag at the C-terminus. This way, the proteins can be purified to check if they are successfully expressed
mKate2 protein with double transcription terminator
Combination of BBa_K1316000 with BBa_K823017
mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli. BBa_K823017 Double Terminator (B0012-B0011)
Belkin laboratory (Belkin et al. Appl Microbiol Biotechnol (2014) 98:885???895)
Promoter of the ybiJ gene, which is activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB.
mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli.
This is a copy of the Part:B0014. Only here the part is cloned in the standard vector pSB1C3 instead of the pSB1AK3.
Double terminator consisting of BBa_B0012 and BBa_B0011
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Conversion of the iGEM parts registry to SBOL2.1