Parts designed and constructed
This part was designed as a ratiometric luciferase reporter. The first promoter, hyperSpank, is LacI - repressed and controls the transcription of a (vibrio harveyi) luxA gene that has been fused at the N-terminus to an mOrange gene via a flexible linker. This was described by Dachuan Ke and Shiao-Chun Tu (2011) as having an additional peak in its emission spectrum at 560 nm, whereas the normal peak is at 490 nm. This is terminated by b0015. Downstream, pVEG controls the translation of the entire normal lux operon, which is again terminated by b0015. The idea is that the normal luciferase output acts as an internal control signal, to which the output of the induced luciferase with the spectral shift can be normalised. We designed this to be compatible with our cheap open-source sensing hardware. This part has some toxicity issues preventing it from being assembled in pSB1C3. We contacted iGEM HQ and were granted exemption from the pSB1C3 standard for this part.
Riboswitch that is highly sensitive to the F- ion. It forms a rho-independent transcriptional terminator in low fluoride concentrations and this structure is removed in the presence of fluoride, allowing transcription. It is sensitive from about 10μM up to around 30mM. Above this concentration the levels of fluoride begin to become toxic and kill the cells. We have characterized this part in three different chassis: TOP10 e.coli, 168 strain bacillus subtilis and a strain of bacillus subtilis with its normal fluoride riboswitch, the crcB gene, knocked out (kindly provided by the Breaker lab in Yale). Results of Miller assays for these three chassis are also provided.
Riboswitch that acts as a regulatory element, truncating transcripts when magnesium is not bound to the RNA. Four Mg2+ binding sites exist, giving this part significantly switch-like behavior. Binding of these sites by Mg2+ results in compaction of the regulatory region of the riboswitch, which in its unbound state acts as an anti-terminator. Loss of this anti-terminator activity results in the activity of a downstream terminator (included in this sequence) which terminates transcription of the gene. May also be affected by other divalent ions, such as manganese.
Riboswitch that acts as a regulatory element, truncating transcripts when magnesium is not bound to the RNA. Four Mg2+ binding sites exist, giving this part significantly switch-like behavior. Binding of these sites by Mg2+ results in compaction of the regulatory region of the riboswitch, which in its unbound state acts as an anti-terminator. Loss of this anti-terminator activity results in the activity of a downstream terminator (included in this sequence) which terminates transcription of the gene. May also be affected by other divalent ions, such as manganese. This version also contains the first eight codons of the first downstream gene in the native Bacillus genome, MgtE. Substitution of the first eight codons of a reporter with these may improve reliability of change of reporter expression.
This part upregulates an operon responsible for germination rate. Bacillus subtilis spores germinate in the presence of L-Alanine. Up-regulation of the spoVA operon increases germination rate in response to L-Alanine. The promoter for the B.subtilis sspB gene (PsspB) is more active than the endogenous spoVA promoter. It is also active during germination. Expression of the spoVA operon under PsspB increases the germination rate. This construct consists mainly of the sspB promoter followed by the first 354bp of the spoVA operon (first 354bp of the spoVAA gene). Since B.subtilis exhibits accurate and efficient homologous recombination, a single cross-over event between the spoVAA region in the BioBrick and the endogenous spoVAA sequence inserts the sspB promoter upstream of the spoVA operon.
Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. The construct that uses an inducible eCFP (E0020) and a constitutively expressed eYFP (E0030) under the control of the constitutive promoter Pveg (K143012). The construct is optimized in both E. coli and B. subtilis, through the use of B. subtilis ribosome binding sites GsiB (K143020) and SpoVG (K143021).
This is the sequence of the non-standard backbone that our luciferase construct, BBa_k911004, was synthesised in. It could not be assembled in the high copy number standard psb1c3 backbone due to toxicity issues. Attempts to assemble the 9kb insert into psb3c5, a low copy number biobrick plasmid have so far been unsuccessful.