Team:Peking/KillingImprovements

Introduction

Algal blooms seriously threat 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 microcystin. Among many kinds of algae 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 out genetically engineered E. coli.

Design

1.Hen egg lysozyme

Microcystis Aeruginosa is a species 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 the 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 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.

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.

All of 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: They are HlyA, HlyB, HlyD and TolC respectively. 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 links 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.

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 RBSes Bba_B0034 before ATG and inserted them at site after the constitutive promoter, Bba_J23105. Then Gibson Assembling was used to assemble these 3 promoter followed by RBS and genes 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 plasmid that mentioned above in the same E. coli BL21(DH3), which was defined as Stain A (Fig. 3).

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(DH3). This strain was defined as Stain B (Fig. 3B). We could further induce both Stain A and Stain 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 [4]. 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].

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

Result

1. Growth of algae and the killing efficiency of hen egg lysozymes

Whether the lysozyme could kill the algae is highly significant for the validity of our design. The growth curve of algae should be measured firstly. OD670nm, the absorbance of Chlorophyll a, was measured to illustrate the algal density. The algae were grinded before measuring OD for higher measurement accuracy. Absorbance was monitored every day until the growth of algae reached a stationary phase (Fig. 1A).

To quantify the killing efficiency of hen egg lysozyme, The killing efficiency was tested by adding different concentration of hen egg lysozymes into algae culture. Since the corpse of dead algae remains float and still have absorbance, we measure the killing effect both by direct observation and absorbance at specific wavelength (Fig. 1B, C).

1. Growth of algae and the killing efficiency of hen egg lysozymes

The result indicates that 200ng/L lysozyme could kill the algae 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 algae.

2.The lysozyme immune 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 (Fig. 2A). To counteract this effect, the best solution to overcome the detrimental effect is to build an immune 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 (Fig. 2B), and further experimental is expected 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. 3). 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.

4.Perspective experiments

Further experiments would focus on testing killing efficiency, testing the lysozyme immune system, and developing the lysozyme secretion system. The construction shown in the design part (LINK) 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 potentially efficiently kill algae while protect the E. coli to maintain productive state.