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PlacIq-LacI-Plac-cI-λPR dual inverter

The dual inverter system consists of PlacIq-LacI-Plac-cI-λPR.

The lacI gene is expressed from an unregulated promoter Placlq, and its product LacI represses the Plac promoter. Likewise, the Plac promoter controls the expression of the cI gene, and its product CI represses the PR promoter. The PR promoter controls the production of the red fluorescent protein (mRFP1), which represents the‘output’for the circuit, whereas the chemical inducer isopropyl β-D-thiogalactopyranoside (IPTG), which binds to LacI tetramers and renders them unable to repress Plac, provides an external control over the network and thus represents the‘input.’

Usage and Biology

The dual inverter consists of lactose operon, CⅠprotein and λPR012. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a brand new composite part.

The LacI repressor is constitutively expressed which inhibits transcription from the pLac promoter in the absence of IPTG, thus λPR012 could be active. While the expression of the CI repressor can be induced by externally added IPTG and inhibits the transcription of λPR012, hence realizing the reversal of the function.

Characterization

In order to characterize PlacIq-LacI-Plac-cI-λPR dual inverter, ECUST_iGEMer constructed a dual-plasmid: pIN1-pIN2.

Figure 0. Gene circuit of inverter system

Before co-expressing this dual-plasmid, we verified the function of pIN1 and pIN2 respectively. pIN1 was composed of PlacIq-LacI-Plac-cI, using GFP(eCFP) as the reporter gene. Since CI and GFP were both under the control of Plac, the expression of GFP(eCFP) was disturbed by CI according to the measurement of GFP(eCFP) fluorescence. However, we performed SDS-PAGE to confirm that the pIN1 exactly worked.

Figure 1. SDS-PAGE results of pIN1

pIN2 was relatively simple, just consisting of λPR012 and using mRFP as the reporter. In the absence of CI protein, λPR012 was constitutively active to express mRFP. So we could clearly recognized the positive colony via observing the color while construction (the red colony might be the positive one). After colony PCR and sanger sequencing, we incubated the positive colony in 5mL M9 containing 0.1% Kan at 37℃ for 12 hr.

Figure 2. Plates of DH5α transformants (pIN2)

Figure 3. pIN2 growing in 5mL LB for12 hr

Figure.3 showed clearly that after 12 hours’ growing in M9, mRFP of pIN2 was highly expressed. In order to characterize the function of pIN2, we set the negative control (pET) and experiment group (pIN2), both growing in 5mL M9 (containing 0.1% Kan) at 37℃ for 12 hr and then being transferred to the secondary culture. We took samples from the secondary culture in 0h, 1h, 2h and 3h and measured the samples’ fluorescence.

Figure 4. Fluorescence intensity of mRFP

The red fluorescence in pIN2 was extremely high and stable, while the red fluorescence of negative control (pET) was barely detectable, conforming that pIN2 worked effectively.

After charactering the pIN1 and pIN2 seperately.The whole circuit PlacIq-LacI-Plac-cI-λpR (pIN1-pIN2) measurement was performed in liquid cultures. pIN1 and pIN2 were initially co-transferred to E.coli DH5α competent cell. The positive colony was then grown in 5mL M9 at 37℃ for 12 hr, then transferred to 100 mL M9 for enlarge cultivation. After the cells growing to OD600=0.5, we added different concentration of IPTG(0.1μM,1μM, 10μM, 100μM, 1000μM ).

Figure 5. Liquid M9 media in 15hr of dual-plasmid (pIN1 + pIN2) in different concentraton of IPTG

We took samples of each IPTG concentration before and after induction (almost 16hr after induction). The samples were then transferred to a 96-well microplate in which mRFP fluorescence (590 nm excitation, 645 nm emission, top 50% cutoff) was measured by using a fluorescence microplate reader. The fluorescence data were normalized against cell densities which were measured by using a microplate reader at 600 nm.

Figure 6. Fluorescence intensity induced by different concentrations of IPTG

Measurements of the circuits in response to varying IPTG levels were summarized in the transfer curve, where each point on the curve represented fluorescence data from three independent cultures of the same circuit under the same induction conditions.

Just as shown in the figure, as the concentration of IPTG rose, the expression of mRFP decreased, indicating that the dual-plasmid worked effectively. The LacI protein was constitutively expressed from PlacIQ and repressed the Plac promoter. Plac transcriptional activity was controlled by modulating the concentration of an externally added inducer, IPTG. The expression of the CI repressor was controlled by Plac . Repressor CI acts on PλRO12 on pIN2 to repress the transcription of the mRFP gene, the output fluorescence indicator. So without IPTG, the CI level was low and mRFP level was high; adding IPTG increased CI levels and in turn decreased mRFP levels. To sum up, ECUST_China iGEMers quantitatively characterized PlacIq-LacI-Plac-cI-λpR dual inverter successfully

References

[1]Yokobayashi Y, Weiss R, Arnold FH. Directed evolution of a genetic circuit. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16587-91. Epub 2002 Nov 25. PubMed PMID: 12451174; PubMed Central PMCID: PMC139187.

Sequence and Features