Team:Worldshaper-Wuhan/Improve
Objective and background
Methods
Result
Conclusion
References
Improvement
Objective and background
BBa_K190019 is a part designed by iGEM Groningen team in 2009. The original purpose of this part is to increase the adsorption capacity of cells to arsenic by overexpressing FMT in E. coli, so as to reducing the pollution of heavy metal arsenic to water. But their results show that the curve of arsenic uptake in E. coli with fMT shows a lower uptake yield than the other strains [1], which means that this part doesn’t work as expected. Therefore, we plan to improve the function of this part to realize their original purpose. based on part BBa_K190019 fMT, is an arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus [2]. OprF is the major outer membrane (OM) protein of Pseudomonas aeruginosa and has been successfully used for presentation of foreign protein on the cell surface [3]. Therefore, our hypothesis is to use the bacterial surface display technology to display fMT on the bacterial surface, and thus enhance the ability of absorbing arsenic.
Methods:
IPTG induction and protein sample preparation
Constructed plasmid pET24a(+)-orpF-linker-fMT and pET24a(+)-fMT was transformed into E.coli BL21(DE3) strain. Single colony was selected to inoculate LB broth containing kanamycin and cultured overnight. Then overnight culture was inoculated in fresh LB broth containing kanamycin at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.6-0.8. IPTG was added to the bacterial liquid for induction, so that the final concentration of IPTG was 1mM,cultured at 25℃ overnight. After induction, the bacteria were centrifuged, and the bacteria pellet was treated with 2XSDS-PAGE loading buffer and boiled for 5-10min for the preparation of protein samples.
SDS-PAGE
A 12% SDS-PAGE gel was prepared, as the expected molecular weight of fMT and oprF-fMT fusion protein were about 10Kda and 34Kda. Then 5μl marker or 30μl protein samples were loaded in each lane. Run SDS-PAGE and the electrophoresis was complete when the the dye front migrates about 2mm from the bottom of the gel. The gel was stained with Coomassie brilliant blue for 15 min, the eluent was used overnight and the results were observed the next day.
Arsenic concentration detection
Collecting the precipitation of the bacterial after all the induced bacteria were centrifuged overnight. After that, using the PBS to wash the bacteria for three times and then centrifuge the it, which can make the dry weight of the two bacteria was adjusted to be consistent as possible. After this, the sample were resuspended with PBS and divided into 15mL centrifuge tubes. Each tube contained 6ml bacterial liquid. As3+ was added to the tubes to make the final concentrations of 5uM. Samples were taken at 2 time points, which were 15min and 45min after adding the arsenic solution, respectively. Finally, removing the impurities by using a 0.22um filter. The concentration of arsenic ions was tested by ICP-MS.
Result
Recombinant plasmid
As shown in the Fig.1, the recombinant plasmid PET24a-oprf-fMT was successfully synthesized.
Fig.1 Plasimd DNA gel
SDS-Page analysis
As shown in the Fig.2, two proteins about 10Kda and 34kda were successfully expressed after IPTG induction, which were consistent with the expected molecular weight of FMT and OPRF-FMT, indicating that FMT protein and OPRF-fMT fusion protein were successfully expressed under the induction of IPTG.
Fig.2 IPTG-induced protein expression in SDS-PAGE. Lane M: protein marker, Lane 1: FMT protein expression without the induction of IPTG, Lane 2: FMT protein expression under the induction of IPTG, Lane 3: OPRF-FMT fusion protein expression without the induction of IPTG, Lane 4: OPRF-FMT expression under the induction of IPTG.
Analysis of arsenic adsorption results
Fig.3 Arsenic accumulation in E. coli cells with part BBa_K190019 and BBa_K3638000 As shown in Fig.3, 15 minutes after adding the arsenic solution, the strains containing FMT protein or OPRF-FMT protein had substantially the same arsenic adsorption efficiency, both of which were less than 1%. 45 minutes after adding the arsenic solution, the arsenic adsorption rate of strain with FMT protein reached 3%, while strain with OPRF-FMT protein reached 9%, which was 3-fold compared with the control group strain with FMT protein.
Conclusion
Compared with the control group, the recombinant strain expressing oprf-fmt has a significantly higher adsorption capacity for arsenic than the control group, which has a better effect than the original part. Compared with part BBa_K190019, the new part we designed improves the arsenic absorption ability of E. coli.
References
[1] http://parts.igem.org/Part:BBa_K190019
[2] S Singh, A Mulchandani, W Chen. Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein. Applied and Environmental Microbiology.2008.
[3] E G Rawling, N L Martin, R.E.W. Hancock. Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF. Infection & Immunity.1995.