rrnB P1-LacI-pLac-mCherry plausible stress sensor
Made by combining BBa_K639002, BBa_K639000. BBa_K639002 is an improved version of BBa_K112118 made by PCR-amplification with BBa_K112118 as template. BBa_K639000 is made solely from existing registry parts.
The stringent response in bacteria is caused by amino-acid starvation, fatty acid limitation, iron limitation, heat shock and other stress conditions. As a response under these conditions, ''in vitro'' studies have suggested that the alarmone guanosine tetraphosphate (ppGpp) increase to modulate transcription to promote survival. The increase in ppGpp levels causes a redirection of transcription so that genes important for survival are favoured at the expense of those required for growth and proliferation [1]. So, could we use ppGpp as signal molecule to find out when cells are stressed? '''The solution''' Our system will be based on a promoter that is important for regulating growth and proliferation. At the moment we are trying to use the ''rrnB-p1'' promoter, which has been shown in earlier studies to be highly regulated by the ppGpp molecule. Hopefully the promoter will be down regulated enough by increased levels of ppGpp to turn the repressor ''lacI'' it controls completely off. The lacI represses a second promoter ''pLac'' that induces the production of red fluorescent protein (mCherry) and turns the cells red. Reference: http://www.sciencedirect.com/science/article/pii/S0966842X05000788 1 Magnusson, L. U., A. Farewell, et al. (2005). "ppGpp: a global regulator in Escherichia coli." Trends Microbiol 13(5): 236-242
LacI-pLac-mCherry with RBS and terminator
This is a composite part and is made only from parts in the registry.
This brick will yield red colonies as it is. When a promoter is inserted in front of the brick, the red color will disappear or be weaker, due to production of LacI that repress lac-promoter. One way of using this brick is to insert a promoter that you suspect, or know is repressed by a molecule or other stimulus. When the promoter is inhibited, transcription of LacI will stop and mCherry will be produced. Thus, the stimuli can be directly measured as intensity of red colour or fluorescense.
BBa_K112118, rrnB P1 promoter BioBrick in BBb format made by the Berkley iGEM team in 2008.
rrnB P1 promoter with standard prefix and suffix in BBa format. This is a modified version of the rrnB P1 promoter BioBrick in BBb format made by the Berkley iGEM team in 2008.
The BioBrick that the Berkeley team made is in BBb format. This means that the BioBrick is flanked by a prefix and a suffix that is different from the ones that are normally used. Therefore, this BioBrick was initially not compatible with the other BioBricks of normal standard (BBa). To deal with this problem, the rrnB P1 promoter was amplified using PCR. The original promoter made by the Berkeley iGEM team was used as template DNA. To make the amplified brick compatible with the rest of our construct, the standard prefix and suffix was added to our primers.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. 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 for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
represillator of Elowitz and Leibler (2000)
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Double terminator consisting of BBa_B0010 and BBa_B0012
Promoter (lacI regulated, lambda pL hybrid)
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
mCherry, bacterial with RBS and forward terminator
Combines BBa_B0015 forward terminator with BBa_J06602 mCherry, bacterial with RBS
Transcriptional terminator consisting of a 64 bp stem-loop.
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.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible
mRFP (DsRed) derived, altered to be a biobrick by removing a PstI site and adding in the ends.
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
Conversion of the iGEM parts registry to SBOL2.1