Team:Peking/Killing

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Introduction

Algal blooms seriously threaten the ecological integrity and sustainability of aquatic ecosystems. They can deplete oxygen causing harmful effects to the phytoplankton, and also produce a variety of toxic secondary metabolites such as microcystins. Among many kinds of cyanobacteria potentially causing water bloom, Microcystis aeruginosa accounts for a significant proportion[1]. We developed a new approach to control the population of Microcystis aeruginosa in the water which can overcome the weakness of other methods. Our genetically engineered E. coli, which can express and secrete hen egg lysozyme and kill Microcystis aeruginosa efficiently, safely, and controllably, with the help of α- hemolysin type I secretion system in E. coli. Moreover, an immunity system is introduced into the E. coli in case that secreted lysozyme could potentially be harmful to our genetically engineered E. coli.

Design

1.Hen egg lysozyme

Microcystis aeruginosa is a kind of freshwater cyanobacteria which can form harmful algal blooms (HABs)[1]. It almost has the same cell wall components with gram negative bacteria, such as outer membrane, peptidoglycan and inner membrane. Peptidoglycan, as an important structural component of bacterial cell wall, can provide resistance against turgor pressure [2]. Peptidoglycan can be cleaved by bacterial cell wall hydrolases (BCWHs), causing the lysis of bacteria. So we put our attention to lysozymes, the well-known and best-studied group of BCWHs.

Among various kinds of lysozymes, we choose to work with hen egg lysozyme. Hen egg lysozyme, also known as lysozyme C (chicken-type), is one of the most widely used lysozyme, which has high antibacterial effect and is easily available. Hen egg lysozyme is a kind of 1,4 -β-N- acetylmuramidase which causes the cleaving of the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the bacterial peptidoglycan, further causing the lysis of bacteria[3]. The hen egg lysozyme gene was de novo synthesized from commercial company (Genscript, Nanjing, China), and we cloned this gene into the plasmid pET-21a(+) to test the efficiency of lysozyme being expressed from engineered E. coli. This plasmid was transformed into E. coli BL21(DE3) and defined as strain A (Fig. 1).

Figure 1. Lysozyme gene was cloned into pET-21a(+). This plasmid was transformed into E. coli BL21(DE3) and defined as strain A.

2.Secretion

To achieve our goal of controlling the growth of Microcystis aeruginosa, our E. coli should have the ability to secrete hen egg lysozyme. Therefore it`s necessary to introduce a secretion system to our E. coli.

To date, five kinds of translocation pathways have been identified in E. coli. These pathways can either deliver proteins from the cytosol to the medium through only one-step process, or via a periplasmic intermediate which need two steps. In order to prevent the contact between lysozyme and peptidoglycan, we utilized a one-step process system, type I secretion system, to deliver the lysozyme to the medium directly.

Type I secretion system, which is also known as ABC transporter, works in a continuous secretion process across both the inner and the outer membrane of gram-negative bacteria. The proteins involved in type I secretion system form a channel that exports proteins from the cytoplasm to the extracellular environment.

Among the type I secretion systems, α-hemolysin(HlyA) secretion system is the best characterized and studied which has been widely used. Therefore we chose to work with this system to achieve the secretion of hen egg lysozyme.

α-hemolysin(HlyA) secretion system contains 4 parts: HlyA, HlyB, HlyD and TolC[4]. HlyA is the C-terminal signal sequence of α-hemolysin, which can be recognized by HlyB. HlyB is an ATP-binding cassette. HlyD is a membrane fusion protein, which can be linked between the outer and the inner membrane components of the system. And TolC is a specific outer membrane protein, which forms a long channel throughout the outer membrane and the periplasm, largely open towards the extracellular medium(Fig. 2(a)).

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Figure 2. Mechanism of outer-membrane-secretion of lysozyme through α-hemolysin secretion system. Lysozyme is directly transported into extracellular space, thus avoid its close contact to cell wall. The application of type I secretion system ensures the engineered E.coli would not be killed during the expression of lysozyme.

We got the following genes, HlyB (Genscript, Nanjing, China), HlyD (Genscript, Nanjing, China) and TolC (BBa_K554009 from iGEM part). We firstly cloned these three genes with RBS BBa_B0034 before ATG and inserted them at site after the constitutive promoter, BBa_J23105. Then the Gibson Assembly was used to assemble these 3 genes with promoter and RBS together.

At the same time, we use pET-21a(+) as a backbone to constructed another plasmid, which contains the hen egg lysozyme gene, a Glu-Ser linker, and a HlyA signal sequence at the C-terminal of hen egg lysozyme-GS linker. We did the co-transformation, and put these two plasmids mentioned above in the same E. coli BL21(DE3), which was defined as Strain A.

a

b

Fiugre 3(a) Genetic circuit for the transporter of α-hemolysin secretion system. HlyB, HlyD and TolC were sequentially assembled together, each followed a promoter and RBS.(b) Circuit designed for outer membrane translocation of hen egg lysozyme. HlyA is the signal sequence for a-hemolysin secretion system, and the GS linker is designed to avoid the steric effect between lysozyme and signal sequence.

In order to improve the efficiency of the hen egg lysozyme secretion, we also constructed a plasmid that contains both the lysozyme-linker-hlyA signal sequence and these 3 components. We transformed this plasmid, whose backbone is pET-21a(+), into the the E. coli BL21(DE3). This strain was defined as Strain B (Fig. 3b). We could further induce both Strain A and Strain B with IPTG and then tested the effect of the lysozyme secretion as well as the killing effect of the secreted lysozyme.

3. Immunity System

The function of lysozyme is to provide hydrolysis of peptidoglycan by lysing bacterial cell wall. Under the critical threat of lysozymes, bacteria in turn evolved mechanisms to avoid bacteriolysis, such as highly specific and potent lysozyme inhibitors production[5]. There are several inhibitors that are specific for the hen egg lysozyme. In our project, we introduced the protein ykfE to protect our E. coli effectively against lysozyme while killing Microcystis aeruginosa with lysozyme. ykfE is the product of the ORFan gene, which is one of the inhibitor of various kinds of lysozyme. The ykfE`s inhibition of lysozyme occurs via a key-lock type of interaction (Fig. 4), without the conformational changes in the lysozyme inhibitor and lysozyme molecules [5].

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Figure 4. The protein ykfE can protect E. coli against lysozyme while killing Microcystis aeruginosa via a key-lock type of interaction.

There are several inhibitors that are specific for the hen egg lysozyme. In our project, we introduced the protein ykfE to protect our E. coli effectively against lysozyme while killing Microcystis aeruginosa with lysozyme. ykfE is the product of the ORFan gene, which is one of the inhibitor of various kinds of lysozyme. The ykfE`s inhibition of lysozyme occurs via a key-lock type of interaction (Fig. 4), without the conformational changes in the lysozyme inhibitor and lysozyme molecules[5].

Our construct containing the ykfE gene under control of T7 promoter in the pET-21a(+) plasmid was designated Strain C. This pET-21a(+) plasmid was transformed into E. coli BL21(DE3), and the resulting strain was designated as ykfE overexpression strain (Fig. 5).

Figure 5.The construction of lysozyme immune system. The ykfE is constitutively expressed to guarantee the survival of genetically engineered E.coli when secreting lysozymes.

Results

1. Growth of cyanobacteria and the killing efficiency of hen egg lysozyme

Whether the lysozyme could kill the cyanobacteria is highly significant for the validity of our design. The growth curve of cyanobacteria should be measured firstly. OD670nm, the absorbance of Chlorophyll a, was measured to illustrate the algal density. The cyanobacteria were grinded before measuring OD for higher measurement accuracy. Absorbance was monitored every day until the growth of cyanobacteria reached a stationary phase (Fig. 6a).

Figure 6. (a) The growth curve of cyanobacteria in lab condition. The absorbance was measured after lysing the cyanobacteria with sonication isolating tamber.

To quantify the killing efficiency of hen egg lysozyme, The killing efficiency was tested by adding different concentration of hen egg lysozymes into cyanobacteria culture. Since the corpse of dead cyanobacteria remains float and still have absorbance, we measure the killing effect both by direct observation and absorbance at specific wavelength (Fig. 6b, 6c).

Figure 6.(b) Cyanobacteria exposed to lysozyme at the concentration of 2, 5, 10, 20, 50, 100, 200, 400, 1000, 2000mg/L respectively. The photographs were taken after 0, 1, 3, 6, 12, 24, 36 hours treatment.
Figure 6.(c)Killing curve of hen egg lysozyme against cyanobacteria, exhibited in graded final concentration of 20, 50, 100, 200, 500, 1000, 2000mg/L.

The result indicates that 200mg/L lysozyme could kill the cyanobacteria effectively within 72h. So the hen egg lysozyme, if could be properly expressed by our genetically engineered E. coli, would be a valid approach to kill the cyanobacteria.

2.The lysozyme immunity system

Considering the working mechanism of lysozyme that cleaving the peptidoglycan of bacterial cell wall can also wound the "manufacturer", our genetically engineered E. coli. Such a detrimental effect to E. coli was firstly measured. To counteract this effect, the best solution to overcome the detrimental effect is to build an immunity system for our genetically engineered E. coli. We utilized ykfE, a native inhibitor of lysozyme of E. coli. The circuit for the strain has been constructed, and further experimental is expected to be coming soon.

3. The killing efficiency of lysozyme expressed by genetically engineered E. coli

We have constructed plasmids to express the hen egg lysozyme under the inducible promoter on the expression vector pET-21a(+). The expression of lysozyme in E. coli was verified by PAGE electrophoresis (Fig. 8). We have verified that the hen egg lysozyme was successfully expressed in our E. coli. Although we have prepared the analysis that to use sonication lysed E. coli or purified protein to test whether the lysozyme is functionally expressed, the difficulty in the experimental protocol and time limit restrict our further trials. More data could probably be shown in our coming oral and poster presentations.

Figure 8. Testing expression of hen egg lysozyme in E.coli. E.coli before and after induction was lysed, then lysation was centrifuged. Lysate,supernatant and sediment were analysis by SDS-PAGE. Column 1，2，3: lysate,supernatant, dissolved sediment after induction. Column 4，5，6: lysate,supernatant ,dissolved sediment before induction. The darkened band circled with red rectangular confirms the expression of hen egg lysozyme which has a molecular weight of 14.6kD after induction. However the indication of the band near 17kDa is still unknown.

4.Perspective experiments

Further experiments would focus on testing killing efficiency, testing the lysozyme immunity system, and developing the lysozyme secretion system. The construction shown in the (design part) should transport the properly expressed lysozyme to the out membrane space of E. coli. Further work would significantly strengthen the proposed while not fully achieved killing system. We believe the full version of our killing system would efficiently kill cyanobacteria while protecting the E. coli to maintain productive state.

References

[1] Merel, S., Walker, D., Chicana, R., Snyder, S., Baurès, E., & Thomas, O. (2013). State of knowledge and concerns on cyanobacterial blooms and cyanotoxins. Environment international, 59, 303-327.

[2] Callewaert, L. and Michiels, C.W. (2010) Lysozymes in the animal kingdom.Journal of biosciences, 35(1), 127-160

[3] Callewaert, L., Van Herreweghe, J. M., Vanderkelen, L., Leysen, S., Voet, A., & Michiels, C. W. (2012). Guards of the great wall: bacterial lysozyme inhibitors. Trends in microbiology, 20(10), 501-510.

[4] P. Delepelaire(2004). Type I secretion in gram-negative bacteria. Biochimica et Biophysica Acta, 1694 (2004) 149– 161.

[5] Deckers, D., Masschalck, B., Aertsen, A., Callewaert, L., Van Tiggelen, C. G. M., Atanassova, M., & Michiels, C. W. (2004). Periplasmic lysozyme inhibitor contributes to lysozyme resistance in Escherichia coli. Cellular and Molecular Life Sciences CMLS, 61(10), 1229-1237.

[6] Monchois, V., Abergel, C., Sturgis, J., Jeudy, S., & Claverie, J. M. (2001). Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme. Journal of Biological Chemistry, 276(21), 18437-18441.

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