Team:Peking/finalexample

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. The killing efficiency of purified Hen egg lysozyme against algae

1.1 Growth Curve of algae

Two strains of Microcystis Aeruginosa were involved in our experiment: one was FACHB (Freshwater Algae Culture Collection at the Institute of Hydrobiology) 1343 and the other one is Pcc(Pasteur culture collection)-7806.

In this experiment, we measured the OD670nm, the absobance of Chlorophyll a, as a reflection of algal density. OD was monitored every day until the growth of algae reached a plateau.

1.2. The killing curve of hen egg lysozyme against algae.

5 hours after lysozyme solution was added into well-distributed algal culture, the aggregation and sedimentation of algae could be observed, as shown in fig 2(A). Different volume of 1mg/ml lysozyme solution was added into algae to reach a graded final concentration. OD 670 was checked every day until it stopped decreasing. Result is shown in fig 2.(B) .

2. The killing efficiency of purified hen egg lysozyme against E. coli

Since the effect of lysozyme on algae is due to its ability to hydrolyze the polysaccharides as indicated in(wenxian) ,The same graded concentration of hen egg lysozyme as described above was added into E. coli, and the absorbance of cell culture at OD 600 to test the sensibility of E. coli to lysozyme..

3. ykfE confers resistence against lysozyme to E. coli

Plasmid contains the gene ykfE was transformed into E. coli. Hen egg lysozyme was added into E. coli Stain C to a final concentration of *** and the absorbance at OD 600 was measured. Result is shown in Fig 5.

Based on the previously mentioned experiment, immunity system was introduced into the engineered E. coli. Similar quantitative experiment was completed and result indicated that ykfE can protect E. coli effectively in the environment of hen egg lysozyme. (Fig. 5)

4. The killing efficiency of Lysozyme being expressed from engineered E. coli

E. coli BL21(DE3) carrying the plasmid pET 21a-lysozyme and E. coli carrying the blank plasmid pET21a as a control were induced with IPTG at the same time using the same protocols as described before. After being broken up and centrifuged, the supernatant was isolated and was analyzed by polyacrylamide gel electrophoresis to verify the existence of lysozyme (Fig 6).

Result that presents here shows hen egg lysozyme positive in supernatant of E. coli equipped with the plasmid pET 21a-lysozyme, compared to control group. Both supernatant was then added into Microcystis Aeruginosa, and the killing efficiency can be seen obviously here (Fig 7).

We thus could claim that the lysozyme expressed from our engineered E. coli has the ability to kill Microcystis Aeruginosa.

5. The killing efficiency of Lysozyme being secreted from engineered E. coli

E. coli BL21(DE3) carrying the plasmid pET 21a-lysozyme-lard as well as pET 21a-ABC transporter was induced with IPTG. E. coli carrying plasmid pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor, E. coli with plasmid pET 21a-lysozyme-lard only as well as well as E. coli with pET 21a-blank were induced with IPTG as control groups at the same time. After being centrifuged, supernatant was isolated and was analyzed by polyacrylamide gel electrophoresis to verify the existence of lysozyme (Fig 8).

Result presents here shows Lysozyme positive in supernatant of induced E. coli with pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor and induced E. coli with pET 21a-lysozyme-lard and pET 21a-ABC transporter, while negative in supernatant of E. coli with pET 21a-lysozyme-lard only, or E. coli with pET 21a-blank.

All supernatant was then added into Microcystis Aeruginosa, and the killing effeciency can be seen here (Fig 9). The decrease of OD760nm of culture with supernatant of induced E. coli with pET 21a-lysozyme-lard, pET 21a-ABC transporter and pET 21a-inhibitor as well as induced E. coli with pET 21a-lysozyme-lard and pET 21a-ABC transporter, is in accordance with PAGE result, showing our engineered E. coli are able to kill algae.