What we need to put in mind as a consideration is – what do we do when we fail to collect some of the E. coli? To solve the problem, we have decided to introduce lysis genes in to the E. coli. The whole procedure for collecting heavy metals from contaminated water can be done at night in the dark. The collection of heavy metals and the E. coli should be finished by the time the sun comes out. Lysis genes will be turned on by red light, leading to the death of E. coli that has leaked out from phototaxis or aggregation (Fig.3). By introducing lysis as a consideration for biosafety, we can avoid biohazard.
1. Phototaxis
1-1. Construct
1-2. How it works
When light is shone, CheA phosphorylates itself, which then phosphorylates CheY. CheY then changes the movement of flagellar, and makes phototaxis happen. In the dark the E. coli swims straight, but when there is light, E. coli starts tumbling and as a result moves as if it is moving away from the light.
1-3. Evaluation
We expressed phototaxis device (BBa_K317028) in E.coli DH5α and evaluated its function. The E. coli was pre-cultured in 3 mL LB medium containing Chloramphenicol at 37˚C until the OD660 reached 0.5. 1 mL of the pre-culture was removed into a tube, where all-trans retinal (f.c. 2 μM) was added. The tubes were wrapped in aluminium foil to avoid light, and were cultured by shaking at 37 ˚C for 2 hours. 10 μL of pre-cultured solution was plotted onto semisolid medium of 0.5% agar and 2 μM all-trans retinal, and incubated overnight under light (fluorescent light) or dark (wrapped in aluminium foil) conditions. As a result, we expect to see that the size of the colony will not change under light condition because the cells will not move, but under dark condition, we expect to see that the colonies grow larger because of the induced movement.
2. Aggregation
2.-1 Construct
2-2. How it works
2-3. Evaluation
The E. coli cells containing Antigen43 gene was pre-cultured in 3 mL LB medium at 37 ˚C for 12 hours. After adjusting its OD595, they were pre-cultured solution were suspended into 100 mL LB medium and cultured at 37 ˚C. Its OD595 was measured every 1 hour until the OD595 reached 2.5, and 3 mL of it was put into a test tube for IPTG induction of Antigen43 expression. After 2 hours of culturing at 37 ˚C, OD595 of the liquid culture 1cm from the surface was measured and plotted against a curve.
3. Lysis
3-1. Construct
3-2. How it works
3-3. Evaluation
Lysis genes with and without antiholin was evaluated to see any leakage of lysis gene expression. An E. coli containing an empty vector as a control, an E. coli containing Lysis genes with antiholin, and an E. coli containing Lysis genes without antiholin were cultured at 37 ˚C and OD660 was measured every 30min. to 1 hour.
To find out if IPTG expression of lysis gene work, using the vectors containing holin and endolysin expressed under lac promoter, and antiholin expressed under a constitutive promoter, we transformed E. coli DH5α. The obtained colony pre-cultured overnight in 2 mL LB and 1 mL of it was cultured in 100 mL LB at 37℃. At OD660, IPTG (f.c. 3 mM) was added to induce the expression of Lysis genes.
4. Results&Discussion
4-1. Phototaxis
The results of growing E. coli with and without the phototaxis genes under dark and light conditions for 21 h at 37 ˚C are shown in Figure 10. The drawn circles on the plate around the colony represents the size when it was first plated. All colonies under all conditions grew larger out of the circle, and unfortunately no difference could be observed. This might be due to the fact that we used fluorescence light instead of only blue light, and the phototaxis expression might not have been induced well enough.
4-2. Aggregation
This is the result of antigen43 expression (fig.11).
The red and orange line represents growth curve of cells containing Antigen43 under PLlacO1, and the green and the blue lines represents growth curve of cells containing no Antigen43 gene. There is a significance difference between cells with and without the aggregation proteins, as the OD595 went down just went down to a third within an hour. The OD595 of the cells for control remained stable. However, we could not see the difference between the IPTG induced cells and non-induced cells. This data could suggest that the expression of Antigen43 is not repressed well enough, and is leaking even without being induced. It might also indicate that aggregation can even happen with a small expression.
4-3. Lysis
This is the result of comparison of lysis gene expression with and without antiholin (Fig.12). The yellow line represents control, vector without lysis gene. The blue line shows lysis gene with antiholin and the red line shows lysis gene without antiholin. As you can see, both blue and red line show lower OD660 than the control, indicating that there might be a leakage of lysis gene expression. However system with antiholin kept a higher OD660 than the system without antiholin. Therefore, we concluded that the inducible lysis system consisting of T4 endolysin and holin, functions better with the presence of antiholn.
This is the result of lysis gene expression with antiholin, induced by IPTG at stationary phase (Fig.13). Both of these cultivation curves indicate E. coli with lysis genes with antiholin. IPTG was not added to the yellow culture, and IPTG (f.c. 3mM) was added to the blue culture. Within 2 hours after addition of IPTG, the OD660 went down by 80%, indicating that the lysis genes were successfully induced by IPTG.
5. Summary
Using light to control E. coli makes our system very easy and efficient, we do not need to add and waste chemicals to control E. coli, but just need to shine a certain strength of light at them. We shine blue light at them when we want them to gather around somewhere, or when we want them to aggregate and sink down, or shine red light when we want to get rid of the E. coli to lyse. Unfortunately we could not see a clear result in phototaxis expression, but if we can improve its experimenting condition such as specifying the light for induction, or finding a way to see the change in size of the colony on plate, we might be able to confirm it next time, and apply it to our metal ion collecting system. However, we were still able to confirm genetic expression of lysis and aggregation by using an inducible lac promoter, so we expect them to function when we substitute them to a light sensitive promoter too. Using light to control E. coli that has collected toxic compounds from the environment to make it move, to aggregate, and lyse, is an ideal, efficient and also an environmentally friendly way for collection in our metal ion collecting system.
6. Reference
[1] Jung et al. (2001) An Archeaeal photosignal-Transducing Module Mediates Phototaxis in Escherichia coli. J. Bacteriol., 183, 6365-6371.
[2] Vishwa et al. (2003) Photostimulation of a Sensory Rhodopsin ll/Htrll/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro. Biochemistry, 42, 13887-13892.
[3] Kjærgaard et al. (2000) Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens. J. Bacteriol., 182 (17) 4789–4796.
[4] Young et al. (2000) Phages will out: strategies of host cell lysis. Trends Microbiol, 8 (3) 120-8.
[5] Tran et al. (2005) Periplasmic Domains Define Holin-Antiholin Interactions in T4 Lysis Inhibition. J. Bacteriol., 187 (19) 6631-6640.
[6] Engebrechi and Silverman (1984) Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. USA., 81 (13) 4154-4158.
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