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Team:Bielefeld-Germany/Outlook
From 2012.igem.org
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Going large-scale?
After having developed a plan for a potential application of our project we now plan to test our system in a real sewage treatment plant under strict safety regulations. Furthermore we want to use a lab-scale waste water system to measure the enzyme activity of our immobilized laccases in active sludge and in a fixed bed reactor under realistic conditions.
Shuttle vector
The shuttle vector (pECPP11JS) <partinfo>BBa_K863204</partinfo> could be constructed. The following step is to do site directed mutagenesis to eliminate the illegal XbaI restriction site in his4 gene. At next the genotype of the P. pastoris mutants have to be characterized as Mut+ or Muts transformants via PCR with 5AOX-Genotype-FW and TT-Genotype-RV primer. Afterwards the GFP::pECPP11JS construct have to sequenced and transformed into P. pastoris cells. The same procedures have to be repeated with the laccases from Trametes versiculor TVEL5, Arabidopsis thalina ATHL and from Pycnoporus cinnabarinus PCIL35.
After successfully site-directed integration of the shuttle vector in P. pastoris GS115 and genotype characterization, a methanol-induced cultivation is following. With an ion exchange chromatography the laccases can be purified and detected with a SDS-PAGE and MALDI-TOF-MS.
Cellulose Binding Domain
Many different BioBricks could be constructed and cloned. Beneath them three different constructs to express reporter proteins, but a detectable fusion-protein with a cellulose binding domain (CBD) and a green fluorescent protein (GFP) to characterize the binding capacity could not be produced. The switching from induced (T7-promoter) to a constitutive promoter did not have an effect on the expression. Future research will focus on the linker between CBDs and the reporter GFP and the order of CBD and GFP. By now a four amino acid long linker is placed between the CBD and the GFP. Primers to expand the linker have arrived, but could not be applied successful. Primers to make it possible to change the order of the CDB and GFP also have been designed and arrived recently, but could not yet been tested.
Cultivation and Purification
Until now, the laccases from E. coli (ECOL), B. pumilis (BPUL), B. halodurans (BHAL) and T. thermophilus (TTHL) were produced and purified. As a first approach a batch cultivation and a quick and dirty purification procedure was chosen. The next steps will be a scale up for the B. halodurans laccase (BHAL) and a screening for cultivation conditions for the laccase from X. campestris in E coli Rosetta-Gami 2.
To improve the production and to produce a higher amount of laccases several screening approaches, like the cultivation in another medium (e.g. HSG-medium) as well as the establishment of a fed-batch cultivation with different feeding strategies to increase the durance of protein expression, are possible . Additional different fermentation condition can be proved to identify the best fermentation parameters like: pO2, agitation control, pH, temperature, air composition, etc.
For an improvement of the purification various possibilities exist. During our research the laccases were captured by a Ni-NTA resin and eluted with two elution strategies. To enhance the effectiveness of the purification procedure different elution strategies could be screened with varying elution gradients. The purification of the laccases can be expanded by 2 or 3 more steps for a better purification and a final polishing. Potential steps could be an Ion Exchange Chromatography with a following Size Exclusion Chromatography. Furthermore different buffers and socalled washbuffers could be tested including different concentration of CuCl2 to prove if this has a positive influence on the purification procedure.
Activity Tests
Four different laccases have been produced via heterologous expression which showed the ability to oxidize ABTS. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 ECOL] activity have been characterized further to gain information about optimal pH or ideal CuCl2 concentration. Still there are some measurements that would help to characterize these laccases in more detail. One important aspect is to determine the optimal pH of our produced laccases. Until now we have tested pH 1, pH 3, pH 5, pH 7 and pH 9. Since waste water in waste water treatment plants has a pH of 6.9 in average we plan to split the range of pH 5 to pH 7 in smaller intervals and test the activity of our laccases with these pHs. Furthermore we have observed a decrease in laccase activity when incubation with 0.7 mM CuCl2 before measurements. Using higher concentrations of CuCl2 should clarify if there is a negative influence on the activity of the laccases after incubation with distinct CuCl2 concentrations. Additionally we tested laccase activity using different amounts of ABTS to calculate KM and Kcat values. These values are going to be defined and included in our model.
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 TTHL] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccases are only tested on positive activity until now. These enzymes are going to be characterized further to be comparable to the laccases already characterized in detail.
Another interesting approach would be the characterization of the stability of our gained enzymes. This would help us to define the duration that our laccases are active in the waste water treatment plants before they have to be exchanged by new active ones.
Immobilization
An immobilization of Trametes versicolor- Laccase (named TVEL0), E. coli BL21- Laccase (CueO named ECOL) and Bacillus pumilus DSM 27 -Laccase (CotA named BPUL) on CPC-beads was successfully achieved. The next step would be to immobilize the laccases Lbh1 from Bacillus halodurans C-125 (named BHAL) and Ltth from Thermus thermophilus HB27 (named TTHL) and develop an optimal immobilization strategy for them. Another aspect would be to consider a method to increase the enzymatic activity of immobilized ECOL, as well as to raise the binding capacity of BPUL to the beads.
Substrate Analytic
Anthracene, lindane and diclofenac are to be detected with the HPLC. Ibuprofen and naproxen need an improved calibration curve. For naphthalene, acenaphthene and phenantrene we have to test different buffers or lower the temperature too keep it more stable. We have only tested BPUL and ECOL so far. "Team Activity Test" showed that TTHL is active so we can test this laccase for the different substrates. Also there will be the Trametis versicolor laccases TVEL5, TVEL10, TVEL13 and TVEL20. Additional we want to determine kcat/km for the degradable substances. On the LC-MS we did not measure our substrates with our self produced laccases. If we get more time granted, we would test our self produced laccases with the substrates. Besides we did not measure all substrates that needed to be measured. Furter we want to identify degradation products.
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