Background of quorum sensing and acyl homoserine lactones
Explanation of the sensing circuit in E. coli and in B. subtilis
Collected results and discussion of our laboratory work
The bidirectional sensing circuit includes two transcriptional units (TU). TU 1 contains the constitutive promoter PT7 and either the genes for the transcription factors QscR, LasR or it contains mkate2. TU 2 contains the inducible promoter PPA1897 or PLux which control the expression of egfp. An overview of all used TUs is shown in Figure 1. The transcriptional units were inserted into pDGB3_alpha vectors via a GoldenBraid reaction. The DNA constructs were ordered at Twist Biosiences. TU 1 was inserted into pDGB3_alpha_1R and TU 2 was inserted into pDGB3_alpha_2.
Following the NEB high efficiency transformation protocol, the GoldenBraid reaction mix was transformed into NEB 10 beta Escherichia coli cells and plated onto agar plates supplemented with kanamycin and X-gal. For each GoldenBraid reaction 50 µL as well as 250 µL of the transformation batch were plated. Then, the agar plates were incubated overnight at 37°C.
Three Colonies from each GoldenBraid reaction were picked to inoculate 4 mL LB10 medium supplemented with 50 µg/mL kanamycin. The cultures were incubated over night at 37°C and 160 rpm.
The alpha plasmids were prepped with the Promega Pure Yield Plasmid Prep Kit using 4 ml of overnight culture. We performed a PCR amplification of an insert-backbone fragment as well as a gel electrophoresis to ensure a correct insertion of the TU into the alpha vector. Additionally, the inserts were sequenced by eurofins.
After the sequencing the success of the GoldenBraid reaction was confirmed. PT7_mkate2 , PT7_qscr were correctly integrated into alpha_1R vectors. The part PPA1897_egfp was correctly integrated into the alpha_2 vector. PLux_egfp has a point mutation from G > A at position 95 in the open reading frame of egfp which after translation leads to eGFP containing the amino acid G > D at position 32. This could result in a loss of fluorescence later on.
These alpha vectors were used to assemble the genetic circuit. Therefore, two transcriptional units were assembled into a pDGB3_omega_1 vector using another GoldenBraid reaction.
Following the NEB high efficiency transformation protocol, the GoldenBraid reaction mix was transformed into NEB 10 beta E. coli cells and plated onto agar plates supplemented with spectinomycin and X-gal. For each GoldenBraid reaction 50 µL as well as 250 µL of the transformation mix were plated. The omega_1 plasmids were prepped with the Monarch Plasmid Miniprep kit using 4 ml of overnight culture. We performed a restriction digest and gel electrophoresis using NdeI to ensure a correct insertion of both TUs into the omega vector. Additionally, the inserts were sequenced by eurofins.
Aligning the sequencing results of mkate2_PPA1897 with the expected omega vector containing the insert showed an alignment with the backbone but none with the insert. Consequently, the GoldenBraid reaction failed. The sequencing of mkate2_PLux aligned perfectly to the expected construct. The sequencing of qscr_PPA1897 confirmed a successful GoldenBraid reaction except for a point mutation inside the PPA1897 promoter at base 1054 from G to T (Figure 2). Aligning the sequencing results of lasR_PLux with the expected omega vector containing the insert showed an alignment with the backbone but not the insert. Consequently, the GoldenBraid reaction failed.
Due to a lack of time in the laboratory we decided to continue working with the two successfully cloned constructs, mKATE2_PLux and QscR_PPA1897.
The fluorescence assay was prepared as described in the Methodic Protocols. For each construct two clones (A and B) were tested. After approximately one hour the production of the T7 polymerase was induced by 0,5 mM IPTG. Therefore, the T7 promoter used in our constructs was activated.
To monitor the IPTG induction, the mKATE2 fluorescence was measured every 45 min (Figure 3). The emission wavelength used was 633 nm, whereas the excitation wavelength used and measured was 588 nm.1
As expected for the construct containing mKATE2, the extinction at 588 nm increased, while for the construct containing QscR no fluorescence could be measured.
We then tried adding 3OC12-HSL to induce the sensing circuit after 60 and 120 minutes to activate the expression of egfp. The 3OC12-HSL concentration was varied between 10-4 mM and 10-8 mM, but no green fluorescence was observed.
This could be due to the mutation in QscR, potentially causing the transcription factor to lose its function. Otherwise, another reason could be due to the design of our construct. We were planning to vary the combination of promoter and RBS, but we started with a combination which was never tested before and this might have not been working as expected. In the future, a combination of promoter, spacer and RBS previously confirmed to be functional should be used as a positive reference.
- 1. Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, et al. Far-red fluorescent tags for protein imaging in living tissues. Biochemical Journal. 2009:567–574. http://dx.doi.org/10.1042/BJ20081949. doi:10.1042/bj20081949