Team:Tokyo-NoKoGen/Project/lysis

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In general, extracting something from E. coli, there are some annoying procedures (e.g., centrifugation, sonication…). To avoid them, we focus on autolysis system. In the autolysis system of T4 phage, holin and endolysin make E. coli lyse. Holin proteins causes "pores" in the inner membrane of E. coli, which allows endolysin to access and break down the peptidoglycan in the periplasm, causing lysis. So, we construct the autolysis device with these lysis proteins for disruption of E. coli. And more, for severe control of autolysis, we designed the trigger of the autolysis is quorum sensing which would be induced by aggregation of E. coli in EcoTanker system. We propose the autolysis device containing holin and endolysin which is easy and controlled method of extracting the EcoTanker.

Autolysis device includes some lysis protein, Holin, Endolysin and Anti-holin. All of these proteins are derived from enterobacteria phage T4. Holin proteins causes "pores" in the inner membrane of E. coli, which allows endolysin to access and break down the peptidoglycan in the periplasm, causing lysis (Fig.1) [1]. Antiholin binds holin and prevents holin multimers from forming pores on the inner membrane of bacteria. When the device is off, higher expression of antiholin compared to holin is required for better stability of the device [2]. We will construct the autolysis device with these lysis proteins from a registry part BBa_K1128808 (Berkeley team (iGEM 2008)).

Quorum sensing, a mechanism present in some microorganisms by which a bacterium can detect the presence of other bacteria in the neighborhood. Quorum sensing is the regulation system of gene expression in response to cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers (AI) that increase in concentration as a cell density (cell aggregation) (Fig.2) [3]. The high concentration of AI after the aggregation of cells in EcoTanker causes the expression of genes for lysis proteins. The regulation seems to be severe control of autolysis. Quorum sensing device include two proteins, LuxI and LuxR derived from V.fischeri. LuxI is Athyl homoserine lactone (AHL; is one of the AI) synthetase and LuxR is AHL receptor. The complex of AHL and AHL receptors induces expression of the downstream genes as lysis device.

How does this device work in EcoTanker?

In EcoTanker system, the lysis and quorum sensing device works with the device cording plasmid shown in Fig. 3.
Lysis device works as disruption of E. coli, and quorum sensing device works as the trigger of the lysis function. After the aggregation step in EcoTanker, the cell-population density and AHL is higher before the step. Then, the complex of AHL and AHL receptor are formed and the downstream genes cording lysis device can be expressed.

Progress

We had already constructed the device cording genes, (A) and (B) shown in Fig.3.

Construction of BBa_K317037-K317039 (Fig.3 (A))

At first, we did PCR.to amplify DNA fragments shown below from BBa_F2621. Then, we confirmed the DNA fragments length by electrophoresis. After that, the purified DNA fragments were ligated with digested pSB1C3 and pSB1A2- Lysis (BBa_K112808) and we did transformation of DH5α with these ligated samples. Plasmid extraction from obtained colonies and sequence analysis was done. Obtained plasmid were registered, BBa_K317037 and BBa_K317038, respectively (Fig.4).



Second, we ligated the digested BBa_J23102 and BBa_K317038 by 3A assembly. And more, we did transformation of DH5α with these ligated samples. Plasmid extraction from obtained colonies and sequence analysis was done. Obtained plasmid were registered BBa_K317039 (Fig.3 (A)).

Construction of BBa_K317042 and BBa_K317040 (Fig.3 (B)) for the characterization and application of BBa_K112808

First, we amplified Plac (BBa_R0010) from RFP coding device (BBa_J04450) by using BBa_G00100 as forward primer and Plac-Rev as reverse primer. Then, digested the PCR product with EcoRI and SpeI and ligated into pSB1C3. Next, construct RBS-lacI-Double terminator-Plac. This was constructed by 3A assembly and used BBa_I732820. After analyzing the sequence, RBS-lacI-Double terminator-Plac was ligated into BBa_J23109, which was digested with SpeI and PstI. We confirmed the construction of BBa_K317042 by sequence analysis.

Second, we ligated the digested BBa_K317042 and BBa_K317038 by 3A assembly. And more, we did transformation of DH5α with these ligated samples. Plasmid extraction from obtained colonies and sequence analysis was done. Obtained plasmid were registered BBa_K317040

Evaluation of BBa_K317039 (Fig.3 (A))

We performed an experiment with our parts above (Fig.3 (A)). The experiment was performed from single colonies in a streaked LB agar plate. At first, we cultured the single colony having the above device in 100 mL LB for 6 hors at 37℃ with agitation. Then, the culture was dispensed into 3 mL scale and AHL (f.c. 50 nM) was added to each dispensed culture to induce the expression of Lysis genes. The obtained culture was incubated for 20 hours at room temperature without agitation. After that, the OD660 of each culture (N=3) was measured. The result was shown below (Fig. 5).

The result showed that the OD660 were different in presence of AHL (red bar) or not (gray bar).
OD660 of samples with the AHL were half less than no additive samples. That indicates that the complex of AHL and Lux activated the expression of lysis genes and the E. coli in the culture were lysed.

Evaluation of BBa_K317040 for the characterization and application of BBa_K112808

We performed an experiment with our parts above (Fig.3 (B)) in order to characterize BBa_K112808 activity. The experiment was performed from single colonies in a streaked LB agar plate. At first, we cultured the single colony having the above device in 100 mL LB for 12 hors at 37℃ with agitation. Then, the culture was dispensed into 3 mL scale and IPTG (f.c. 0.3 mM) was added to each dispensed culture to induce the expression of Lysis genes. The obtained culture was incubated for 20 hours at room temperature without agitation. After that, the OD660 of each culture (N=3) was measured. The result was shown (Fig.6).

The result showed that the OD660 were different in presence of IPTG (orange bar) or not (gray bar). OD660 of samples with the IPTG were half less than no additive samples. That indicates IPTG induced the expression of lysis genes and the E. coli in the culture were lysed.

Conclusion and perspective