Team:Hong Kong-CUHK/Description
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Part Improvement:
Background:
Magnetosomes, an organelle having encapsulating magnetic iron crystal can be applied in many different aspects. One of the various applications is to construct a more efficient microbial fuel cell (MFC). MFC is a machine which uses electrons produced by microorganism to generate electricity. If we genetically modify the bacteria Azotobacter vinelandii to have magnetosomes, magnetosomes inside them would be attracted towards the electrodes by magnetic force and in the process, bringing the whole bacteria along with it. In consequence, the physical distance between the bacteria and electrodes will be shortened. As a result, this will increase the efficiency of the MFC as the diffusion rate for the electron to the electrode can be greatly increased.
Additionally, in the review of the previous iGEM teams, the idea of constructing an MFC has been popular. For example, the iGEM 2013 Bielefeld-Germany team also constructed an MFC. After a brief study of their project, we understood that one of their manipulated parts is the oprF gene, which has the biobrick code - k1172501. The team has claimed that oprF, an outer membrane porin, could increase the efficiency of MFC by allowing electron shuttle-mediated extracellular electron transfer from bacteria to electrodes.
Investigation on K1172501
However, with a further study, we have found that the translated sequence exists many stop codons in all of the wrong places. Moreover, with a careful checking of the sequence in silico, it is concluded that the sequence of the biobrick - k1172501 provided by the Bielefeld-Germany team will not be able to translate into an oprF porin protein. As the DNA sequences of K1172501 is greatly different from oprF DNA sequence from Pseudomonas fluorescens which is the bacteria Germany team claimed to obtain oprF gene sequence.
OprF in Azotobacter vinelandii
We have found that OprF exists on the outer membrane of Azotobacter vinelandii, which is the bacteria we have been working on. Therefore we chose the Azotobacter vinelandii to provide an alternative source of OprF. The sequence provided by theAzotobacter vinelandii can be completely translated to form OprF with no stop codon appearing in the gene excepting in the last residue. For this, we provide the biobrick, BBa_K1648045 and we are planning to provide BBa_K1648047 for insertion of different promoter.
Figure 1. The photo of 1% agarose gel electrophoresis. L: DNA ladder. Lane 1: PCR product of oprF encoding fromthe Azotobactervinelandii strain DJ genome.
Figure 2. Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: Recombination Template for pSB1C3-oprF(BBa_K1648045) with double digestion cut at EcoR1 and PstI sites, with single digestion at Pst1 site, without digestion.
Mutated oprF with Higher Efficiency
Furthermore, to construct a more efficient MFC, a mutated OprF with 5-point mutations is suggested. According to a paper concerning the factors affecting the conformation of OprF, we found that mutation on four Cysteine to four Serine and Lysine to Glycine at 189th position (K189G; C201S; C210S; C216S; C230S) of Azotobacter vinelandii oprF will give higher possibility of open-channel conformation with high porin activity by 5 folds.[2] With the insertion of this mutated OprF into the bacteria, the possibly of electron carrier transmission between bacteria and extracellular as well as the efficiency of MFC would be increased by five folds. Knowing E.coil is capable to form porin using plasmid DNA[1] , we used it to carry out the investigation on the oprF efficiency comparing K1172501, oprF from A. vinelandii and mutated OprF. For this, we are planning to provide the biobrick, BBa_K1648046.
Characterization for different oprF
For comparison, identical promoter, J13002 which is a constitutive promoter, is chosen to add before the gene in pSB1C3 for constitutive expression of different oprF in bacteria. The transformed bacteria with these three plasmids respectively is cultured for the characterization. There are BBa_K1648048 for oprF from Azotobacter vinelandii, BBa_K1648049 for mutated oprF from Azotobacter vinelandii and BBa_K1648050 for BBa_k1172501 from Germany iGEM team.
J13002-oprF(BBa_K1648048):
Figure 3. Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: Recombination Template for J13002-oprF(BBa_K1648048) with double digestion cut at EcoR1 and PstI sites, with single digestion at Pst1 site, without digestion.
R0040-oprF*(BBa_K1648049):
Experiment Set-up and Ongoing Test
In the experiment, we will use the colour change of methylene blue as an indicator to compare the efficiency between the transformed bacteria with different oprF plasmids and wild type bacteria. The desired function of the oprF porin protein for this experiment is to allow the diffusion of (reduced) electron carriers in and out of the periplasmic membrane from the outside of the cell. As the electron carrier (e.g. NAD+) picks up an electron in the periplasmic space (i.e. being reduced to NADH) and diffuse out of the cell through the porin protein; the electron on the NADH will transfer to Methylene Blue (the mediator solution outside the cell). When the Methylene Blue gets reduced to form Leucomethylene Blue, it will have a colour change from the original blue colour to colourless. Hence, the rate of transmission of electron carrier is calculated by the rate of reduction of methylene blue. The experiment is planned to carry out soon. The plasmids will also be transformed into Azotobacter vinelandii for the construction of our MFC.
Reference
1. Sugawara, Etsuko, Keiji Nagano, and Hiroshi Nikaido. "Factors affecting the folding of Pseudomonas aeruginosa OprFporin into the one-domain open conformer." MBio 1.4 (2010): e00228-10.
2. Yong, Yang‐Chun, et al. "Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin." Biotechnology and bioengineering110.2 (2013): 408-416.
