Team:XJTU-China/Contribution
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XJTU-China
Contribution
New documentation to the part, MazF(BBa_K302033)
This year, our team designed a suicide switch mediated by arabinose operon for biosafety. We use MazF to kill the bacteria. We have supplemented the relevant information of this element in the iGEM Parts and added test and characterization to it.
Our MazF comes from the genome of Bacillus Subtilis. By regulating the expression of MazF, we inhibit the life of bacteria when needed to prevent the possible strains and genetic leakage。
MazF is a kind of mRNA interferas. Specifically, it specifically cleaves five-base U^ACAU sequence (^indicating the cleavage site) on RNA[1]. MazF achieves its catalytic function by forming a special MazF-RNA complex. One molecule of 9-mer RNA is bound to one MazF dimer, and two subunits of MazF form the dimer related by local 2-fold symmetry.The RNA in an extended alignment is bound along the RNA binding interface between subunits of the MazF dimer, covering part of this dimeric interface, and MazF interacts extensively with the pentad target sequence present in the RNA chain by interactions with bases[2]. Therefore, the backbone phosphate moieties project outward and away from the protein surface. In this way, scissile phosphate will be attacked by other side chain groups, which causes breaking of P-O bond.
What’s more, it is an important part of Bacillus Subtilis toxin-antitoxin system. Which is essential for the programmed cell death of this developmental bacterium. In normally growing cells, MazF forms a stable complex with its cognate antitoxin, MazE, however, under stress conditions, unstable MazE is preferentially degraded to release free MazF in the cells, which then cleaves cellular mRNAs to inhibit protein synthesis, leading to growth arrest. The protein-protein interaction interface between MazE and MazF (2,843 Å2) is larger than the MazF-RNA interaction interface (2,153 Å2), suggesting that MazF is likely to have a higher affinity for MazE over its RNA substrate. In the B.S MazE-MazF complex structure, MazE binds to MazF along the dimer interface, and it even occupies part of the putative modeled RNA binding site on the second subunit of MazF[3].Thus, in the presence of MazE, MazF cannot bind to or cleave substrate RNA.
Figure 1 the structure of MazF
Figure 2 Material eluted, acetone precipitated and reapplied to an SDS-20% polyacrylamide gel and then silver stained. Slice numbers are the same as in A. E is whole cell extract used as starting material for protein elution. Arrow shows the estimated position of MazF migration.
Biochemical properties of MazF
The size of MazF is about14 kDa. The enzyme activity showed very little pH dependence between pH 5.0 and 9.0 in either 10 mM T ris-maleate or 10 mM citrate-phosphate buffer. It was also active over a wide range of temperatures from 4℃ to 50℃,with an optimum of around 28℃. The enzyme is inhibited by high salt concentrations and optimal enzyme activity was seen in the complete absence of any
divalent cation[4].
Characterization
We transferred the plasmid pBAD-MazF into E.coli DH5α. The bacteria in the experimental group were cultured in the liquid LB-ampicillin medium supplemented with L-arabinose. The concentration of L-arabinose is 1.5μM/L. The bacteria liquid was then diluted and spread on solid medium. 24 hours later, no obvious colony was observed in the group with L-arabinose, but the bacteria in the blank control group grew normally. It is proved that the growth of bacteria is inhibited in the case of MazF expression.
Figure 3 Experimental medium. 6-1 is blank control ,6-4 is experimental group.
New documentation to the part, galU(BBa_K861140)
We have also updated the functions and parameters of the E.coli component. In engineering bacteria, E. coli is expressed in large quantities. The E.coli-galU gene will promote the expression of bacterial extracellular polysaccharides.
The mRNA level of B.S GalU is ~2.4 fold higher than that of E.coli, indicating the significant improvement of enzyme expression and acitivity. However, due to the lower expression of another key enzyme PGM for PS production in BE strain ，the finally EPS yield is lower than that of EE strian, but still comparable.
Measurement
This year, we have made some summaries and improvements on the detection methods of polysaccharide products and common soil components.
We have developed the general method for the determination of extracellular polysaccharide content in the culture medium. We have also designed quantitative methods for the determination of available phosphorus content, total nitrogen content, organic matter content, and various inorganic salt ion content in the desert soil. For the determination of polysaccharide content, the method that uniformly converted the polysaccharide in culture medium into glucose equivalent was developed, followed by the phenol sulfuric acid method that used to determine glucose content. The method will not only ensure the accuracy, but also avoided the tedious operation of polysaccharide purification, multiple separation, hydrolysis and re-separation, which is effective to determine the ability of engineering bacteria producing extracellular polysaccharides. For the determination of soil-related parameters, we have integrated a variety of methods, such as the colorimetric method of chromogenic agent, to determine the content of available phosphorus and other parameters of soil samples collected from several desert areas such as Yulin Cherishing sand plant protection base, which basically proves the feasibility of the method.
Parts library
As there are very few iGEM teams in northwest China, it is very inconvenient to seek help from nearby teams. Based on this, at the Shaanxi meetup in 2019, we decided to establish a northwest database of common genes or parts of iGEM teams in northwest China for the convenience of future teams. This year we will continue to complete the five-year Northwest Alliance plan that we introduced last year. IGEM officials will provide free 20 KB-DNA synthesis per team per year, which we plan to use to build our parts library. This year, we continued to synthesize gene fragments of interest to form a DNA database about 120 KB in length. In addition, we will continue to enrich the database over the next few years, and the part library we built will be free and open to all iGEM teams to benefit teams in the northwest and across the world.
Hardware
In terms of hardware, based on the experimental background of our project, we have produced a hardware called DCSC (Desert climate simulation cabinet) which can precisely simulate the desert environment for the cultivation of our cyanobacteria and symbiotic system. We applied 3D printing technology to produce a complex component in the production steps in the form of a video for the follow-up team to refer to.
Reference：
[1]Park, Jung-Ho, Yamaguchi, Yoshihiro and Inouye, Masayori(2011), Bacillus subtilis MazF‐bs (EndoA) is a UACAU‐specific mRNA interferase, FEBS Letters, 585, doi: 10.1016/j.febslet.2011.07.008
[2]Dhirendra K. Simanshu, Yoshihiro Yamaguchi, Jung-Ho Park, et al. Structural Basis of mRNA Recognition and Cleavage by Toxin MazF and Its Regulation by Antitoxin MazE in Bacillus subtilis. 2013, 52(3):447-458
[3]Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol.
2005; 56:845–857. [PubMed: 15853875]
[4] Pellegrini O, Mathy N, Gogos A, Shapiro L, Condon C. The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. Mol Microbiol. 2005 Jun;56(5):1139-48. doi: 10.1111/j.1365-2958.2005.04606.x. PMID: 15882409.