Difference between revisions of "Part:BBa K1541017"

Revision as of 10:34, 27 October 2014

rhlI with optimized RBS

N-butyryl-L-Homoserine lactone or short C4-HSL

This part contains the part C0170 (RhlI), which is an autoinducer synthesis protein that produces N-butyryl-L-HSL (C4-HSL) which binds to the regulator RhlR (C0171), obtained from Pseudomonas aeruginosa. Since this part was used in a low-copy vector, RBS engineering was performed to increase expression levels of this enzyme and thus also improve the production of C4-HSL.

Usage and Biology

Quorum Sensing

RhlI is the autoinducer synthetase from Pseudomonas aeruginosa, which can synthesize the molecule N-butyryl-L-HSL (C4-HSL). C4-HSL can bind to the regulatory protein RhlR (C0171). Once C4-HSL is bound to RhlR it can activate the promoters R0071 or I14017.

RBS Engineering

The ribosomal binding site (RBS) is a sequence on the mRNA crucial for the translation initiation step in prokaryotes, which highly influences protein levels. The influence of the RBS sequence also depend of the genetic context and identical RBS sequences in different contexts can result in different protein expression levels (also see the Design page for our genetic context). The [http://salis.psu.edu/software/RBSLibraryCalculatorSearchMode RBS Calculater] provided by the Salis Lab was used to find an optimized RBS for the genetic context of rhlI. The calculator provides a value for the translation initiation rate (TIR), which was shown to highly correlate with protein expression levels^{[http://www.nature.com/nbt/journal/v27/n10/full/nbt.1568.html [1]]}.

This optimized RBS was compared to the case where the RBS B0034 is placed upstream of rhlI (compare I9026). The TIR for rhlI with upstream placed B0034 was calculated as approximately 1500 while the optimized construct reaches a TIR of approx. 684200, which is an increase of more than 450 times.

Experimental Validation

Figure 1 Improved RBS performance for rhlI demonstrated with a C4-HSL sensor. The fluorescence per OD₆₀₀ is shown for the plasmid combination containing the Rhl sensor system (pRhl (BBa_I14017) and RhlR (BBa_C0171)) together with the C4-HSL producer RhlI (BBa_C01700). The combination containing the here analyzed part BBa_K1541017 with the rhlI optimized RBS is compared to the case where rhlI is preceded by the RBS BBa_B0034. Additionally these two cases were also measured when C4-HSL was added directly to a final concentration of 1 mM. Data points are mean values of triplicate measurements in 96-well microtiter plates 8 hours after inoculation ± standard deviation.

Figure 2 Improved RBS performance for rhlI demonstrated with a C4-HSL sensor over time. The fluorescence per OD₆₀₀ over time is shown for the plasmid combination containing the Rhl sensor system (pRhl (BBa_I14017) and RhlR (BBa_C0171)) together with the C4-HSL producer RhlI (BBa_C01700). The combination containing the here analyzed part BBa_K1541017 with the rhlI optimized RBS is compared to the case where rhlI is preceded by the RBS BBa_B0034. The timespan shown here starts at the beginning of the exponential growth phase. The end of the exponential growth phase is indicated by a dashed line. Data points are mean values of triplicate measurements in 96-well microtiter ± standard deviation.

Setup

We used an E. coli TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell) and one low copy plasmid (approx. 5 copies). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter pRhl (BBa_I14017), a RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). The second plasmid contained the pBR322 origin (pMB1), an ampicillin resistance gene, and a constitutive promoter BBa_J23100 chosen from the Anderson promoter collection followed by rhlR (BBa_C0171). The first two plasmids are hereafter described as C4-HSL sensor. The third plasmid contained the pBBR1 origin of replication, a tetracycline resistance gene, and for the production of C4-HSL the constitutive promoter BBa_J23100 followed by the here characterized BioBrick BBa_K1541017. As reference another tranformant contained in the third plasmid instead of BBa_K1541017 the RBS BBa_B0034 followed by ’’rhlI’’ (BBa_C0170) as C4-HSL production unit.

The above described E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL), ampicillin (200 μg/mL), and tetracycline (10 μg/mL) to an OD₆₀₀ of about 1.5 (37 °C, 220 rpm). The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in triplicates in microtiter plate format on 96-well plates (200 μL culture volume) for 15 h at 37 °C with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm). For one triplicate of each strain we directly added C4-HSL to a final concentration of 1 mM, the other triplicate of each strain was cultivated without additional C4-HSL. The induction scheme was as follows:

• C4-HSL sensor + B0034-C0170
• C4-HSL sensor + K1541017
• C4-HSL sensor + B0034-C0170 + 1 mM C4-HSL
• C4-HSL sensor + K1541017 + 1 mM C4-HSL

From the the obtained kinetic data, we calculated mean values and plotted a bar plot for 8 hours past inoculation for all the four combinations (Figure 1). In Figure 2 the kinetic fluorescence data is shown for the two first cases from the beginning of the exponential growth phase starting 5 hours past inoculation. The beginning of the stationary growth phase is indicated with a dashed line.

Conclusions

Figure 1 shows that the optimized RBS for rhlI increases the response of the C4-HSL sensor construct compared to the case where the RBS BBa_B034, which is commonly used as strong RBS, precedes the gene for RhlI, the C4-HSL synthetase. The response is even stronger than in the case where C4-HSL is added additionally. In Figure 2 it can also be observed that the presumably increased RhlI expression level leads to a faster response of the C4-HSL sensor indicating that the production rate of C4-HSL could be increased by the higher enzyme level.

References

[1] [http://www.nature.com/nbt/journal/v27/n10/full/nbt.1568.html Salis, H. M., Mirsky, E. A., Voigt, C. A., Automated design of synthetic ribosome binding sites to control protein expression, Nature Biotechnology, 27, 2009]

Sequence and Features