As for the design of intracellular compartment, it is divided into two parts, artificial gene cluster and natural gene cluster mamAB from Magnetospirillum Magneticum AMB-1 strain.

1. Why magnetosome?
      It is generally considered that intracellular membrane structures are unique to eukaryotes, but rare in bacteria. The magnetosome structure of M.magneticum has obvious similarities to eukaryote membranous organelles. Similar to organelles of prokaryotes, magnetosome is packaged by 3 ~ 4 nm thick biofilms, which resemble plasma membrane. Its inner diameter is 10 to 120nm, arranged in chains in wild-type strain. Due to the barrier effect of the MMB, magnetosome membrane cavity is a nanoscale reactor, for the formation of Fe3O4 (iron oxide) requires strict pH environment.
      So, we plan to construct the magnetosome membrane compartment in E.coli as a reactor for future work.


Figure 1.Three-dimensional organization of magnetosomes. An ECT reconstruction of Magnetospirillummagneticum sp.AMB-1. Image courtesy of Zhuo Li & Grant Jensen[3]

2. What is mamAB?
      The construction of magnetosome is guided by a 100Kb long gene sequence which is formed by 14 regions, called magnetosome island (MAI). According to gene knock-out experiments, we know that natural R6, R7, R8, R9 dominant negative the artificial R1, R2, R3, R4, R6, R9, R10, R11, R12, R13, R14 dominant negative contain normal magentosome compartment chain, while magnetism of the R2, R3,R3, R6, R9, R11, R12, R13, R14 dominant negative reduce varying degrees, and only when R5 region (also called mamAB) is knocked out, the magnetosome can’t exist anymore.
      Above all, R5 is required for the construction of magnetosome. That’s why we have hopes in constructing the mamAB gene cluster in E.coli according to homologous recombination. This is what we wish to achieve in the future.


Figure 2.Genomic organization of the MAI and the mamAB gene cluster in Magnetospirillum magneticum AMB-1.[13]

3. How did we design the artificial gene cluster?
      The genes related to the formation of magnetosome are mainly concentrated in MAI, which contains a large number of repeats and antisense gene fragments. But, as we know, because it contains too many regulate sequences; it can mutate easily when oxygen concentration fluctuates. What’s more, it will also be much more difficult to transform such a long sequence into cell. So we decided to transform a plasmid that contains only necessary genes into E.coli to explore the necessary and sufficient condition of MMB formation.
      Magnetosomes are formed in three steps. First, mamK lead the membrane invagination derived from the inner membrane, and magnetosome proteins sorted away from cell membrane proteins. Second, individual invaginations assembly into a chain with the help of MamJ and MamK proteins. Third, iron is transformed into highly ordered magnetite crystals within the magnetosome membrane. In this step, there are many special magnetosome protein such as mamI, mamL, mamB and mamQ assembled onto the membrane while playing an important role on the construction of MMB.
      Two separate models can be envisioned for the role of MamK in organizing the chain.
      It is assumed that MamK interacts to establish the chain by bring newly formed magnetosomes into the magnetosome chain. Alternatively, MamK maintains the magnetosome chain perhaps by preventing the insertion of newly synthesized membrane lipids in the magnetosome chain.
      This model can be envisioned for the role of MamK in organizing the chain. Which is assumed that MamK interacts to establish the chain by bring newly formed magnetosomes into themagnetosome chain. Alternatively, MamK maintains the magnetosome chain perhaps bypreventing the insertion of newly synthesized membrane lipids in the magnetosomechain.


Figure 6.Model for magnetosome formation Magnetosomes are formed in three steps.[14]

      Above all, we designed an artificial gene cluster in order to construct the compartment we need, which includes mamI, mamL, mamB, mamQ and mamK.


Figure 7.The design of artificial magnetosome gene cluster.

4. What is the point of applying MamC::GFP fusion protein?
      MamC is the mostly expressed protein among all the MMB associated proteins, and its structurefits to be an anchorprotein. In our project we combine MamC and GFP as a fusion protein and express it on PSB1C3 vector to show the existence of our intracellular compartment under the fluorescent microscope.


Figure 8.The MamC::GFP expression vector we use to shows the existence of our artificial organelle under the fluorescent microscope.