Part:BBa_K516132:Experience
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Applications of BBa_K516132
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UNIQaea34bcb91d63d8b-partinfo-00000000-QINU UNIQaea34bcb91d63d8b-partinfo-00000001-QINU
SCU-China 2018:
Experiment conditions:
1. 37℃ incubator for E.coli DH5α strain;
2. LB medium with chloramphenicol.
Analyze & Results:
We use this part BBa_K516132 as our original source of RFP proteins. As our expectation, this part can work normally under our experiment conditions and also the part BBa_K2611003 that we added a spacer sequence and NGG site can growth well (as the Figure 1 shows).
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For the improvement, just as Figure 3 shows, the expression of the mRFP increased after the modification. And we can infer that the dCas9 protein can also have a good performance according to the repression of GFP (BBa_K2611001).
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UNIQaea34bcb91d63d8b-partinfo-00000002-QINU
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UNIPV-Pavia iGEM 2011 |
NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5.
Used to quantify promoter strength (relative to BBa_J23101 with RBS B0034) and the RBS efficiency.
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in 'data analysis' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. Salis et al. [Nat Biotec, 2009] stated that 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels' and again 'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.' For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.
On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.
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