We used the biobrick (BBa_E0840) available in the database of the iGEM since 2004 to be connected to our ???essential biobrick??? (BBa_K1355001) and - together - form a system capable of biodetect mercury in bacteria. In the presence of mercury, The MerR protein will bind to it and MerT, MerP and the Green Fluorescent Protein (GFP) protein will be expressed and the biodetection will begin! The MerP and MerT protein are responsible for the transport of mercury from the periplasm to the cytoplasm and the GFP will act as rep??rter to alerting the presence of mercury in the middle and the higher concentration of mercury is, higher will be the GFP expression. And If there is no mercury in the middle, the synthesis of MerT, MerP and GFP protein will be repressed by the MerR repressor protein.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the GFP Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for GFP Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of GFP gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + GFP Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +GFP Biobrick ;
Regulation and transport of mercury ions
This construction is based on sequences present in the O26-CRL plasmid found in Escherichia coli O26. We added a reverse tryptophan operon terminator to ensure transcription termination of the merR messenger RNA. However we didn???t add transcription terminator after the merP gene, aiming connect this biobrick with others, enabling various functions related to mercury
We call this part an ???Essential biobrick??? designed to be the key piece of any genetic construction related to mercury, targeting both a biosensor as a bioaccumulative. This biobrick has bidirectional promoter, the regulator MerR with a terminator reverse from the tryptophan operon and a forward MerP and MerT transporters.
If is desirable to accumulate mercury, assemble this biobrick with a good RBS, a coding sequence of a peptide having affinity to metal and a terminator! Its function will be regulated by presence of mercury through MerR regulator. The bacteria will carry the mercury to cytoplasm until it is linked to the peptide having affinity to the metal, making a inactive mercury. Is not it amazing? This is the strategy that we use in our project! The so called ???Essential Biobrick??? was connected to biobrick for bioremediation, bioaccumulation and bio-detection!
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Conversion of the iGEM parts registry to SBOL2.1