Part:BBa_K516132:Experience

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UNIQcbad2b1ba6ceccf8-partinfo-00000000-QINU UNIQcbad2b1ba6ceccf8-partinfo-00000001-QINU

Characterization

••••• iGEM2019[http://2019.igem.org/Team:BIT# BIT]

iGEM2019 [http://2019.igem.org/Team:BIT# BIT] Expression of BBa-K516132 in microfluidic chip

•••••

In order to verify the possibility of the growth of E. coli and the expression of fluorescent protein in microfluidic chip made of PMMA, we built the K516132 line and transferred it into E. coli. In the cultivating chamber of the microfluidic chip, set the cultivation system as 100 μL, including 100 μL LB medium and 1 μL chloramphenicol. The inoculation quantity of E. coli cultured overnight was 2%, and no e. coli was added to the control group. After the sample addition was completed, transparent tape was used to seal the inlet and outlet of the sample to prevent evaporation from the culture medium in the shaking table. Microfluidic chip was placed under an inverted fluorescence microscope to observe the culture situation and record the growth status of E. coli at this time as 0 hour. Then the microfluidic chip was fixed in a petri dish, and the petri dish was placed in a constant temperature shaking table at 37℃ for culture. The microfluidic chip was taken out every 2 hours and observed under an inverted fluorescence microscope to record the growth of E. coli. The experimental results are shown as follows: The Image J software was used to process the two groups of pictures, and the fluorescence intensity was calculated through gray analysis. The change curve of fluorescence value with time was obtained. The experimental results are shown as follows: It can be seen from the figure, the fluorescence intensity of red fluorescent protein increased significantly with the increase of culture time.

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).

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). UNIQcbad2b1ba6ceccf8-partinfo-00000006-QINU

••••• 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. Characterized together with: BBa_J23101 J23101-RBS34-mRFP-TT BBa_K516130 J23101-RBS30-mRFP-TT BBa_K516131 J23101-RBS31-mRFP-TT 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. NB: in the RPU computation, the J23101-RBS34-mRFP-TT (BBa_J23101) in low copy plasmid pSB4C5 construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. Data are provided as average [Standard error]. J23101 promoter with RBS Scell R.P.U.s RBS30 122 [13.23] 2.45 [0.27] RBS31 2 [0.25] 0.04 [0.01] RBS32 20 [1.7] 0.40 [0.03] RBS34 50 [1.87] 1 [0.02] 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.

===Characterization===