Journal

Overview Prologue

The iGEM-Team Bielefeld 2013 started working and decided to deal with the project 'Microbial Fuel Cell'.

Initiating labwork on the sub-project endogenous mediator Glycerol dehydrogenase.
Starting lab work with the first successful PCR's. The main work however is still in the planning of our project. Designing experiments and a lot of research.
Finding sponsors goes ahead. Many companies like our project and want to support us.

Starting labwork on the sub-project Porins.
Successful PCR on OprF gene from Pseudomonas fluorescens. OprF with Pre- and Suffix overlaps could be amplified from genome.
Planning of our Human Practice projects started and the first participations are fixed.

Poster presentation at the congress ‘Next generation of biotechnological processes 2020+’ in Berlin.
Sponsoring acquisition is nearly finished.
Problem with cloning delaying the progress in the laboratory.
We have published our first press release with a great response. The press release was picked up worldwide.
Starting labwork on the biosafety project.

Our great expert Dr. Falk Harnisch has answered numerous questions about our project and helped us very well.
GldA BioBrick (<bbpart>BBa_K1172201</bbpart>) was examined.
- GldA Biobrick Devices with different promotors and RBS could be added: (<bbpart>BBa_K1172203</bbpart>, <bbpart>BBa_K1172204</bbpart>, <bbpart>BBa_K1172205</bbpart>)
OprF BioBrick (<bbpart>BBa_K1172501</bbpart>) is available.
- OprF Biobrick was functionalized with different promotors and RBS: (<bbpart>BBa_K1172502</bbpart>, <bbpart>BBa_K1172503</bbpart>, <bbpart>BBa_K1172504</bbpart>, <bbpart>BBa_K1172505</bbpart>, <bbpart>BBa_K1172507</bbpart>)
Successful protein expression and overproduction of glycerol dehydrogenase. SDS-PAGE shows GldA at expected size.
We have produced a knockout. This AraC-Deletion in K12 Δalr Δdadx causes that this K12 E.coli can't produce AraC of itself and that it is dependent of the supplementation of D-alanine or the supplementation of a vector where araC is expressed.
We started the wet-lab work on riboflavins by isolating the rib-gene-cluster form the genomic sequence of shewanelle oneidensis

Successful characterization of Glycerol dehydrogenase
- NADH-Assays show an increasing intra- and extracellular NADH concentration for Escherichia coli KRX with overexpression of glycerol dehydrogenase.
- Glycerol dehydrogenase was examined by MALDI-TOF MS/MS with a Mascot Score of 266 against Escherichia coli database.

Great characterization of outer membrane porin OprF
- Successful Hexadecan-Assay for characterization of hydrophobicity of the outer membrane. The hydrophobicity increases continuously with increasing promoter strength up to 221% hydrophobizity in contrast to the wild type.
- ONPG and NPN uptake assays were performed. Membrane permeability is continuously increasing for Escherichia coli with heterologeous expression of OprF.
- According to AFM images, E. coli KRX with OprF shows a slightly rougher cell surface morphology in contrast to Escherichia coli KRX wild type, which confirms the results.
- We can see a strong overexpression band on SDS-PAGE at the expected OprF size of about 36 kDa which is equated with a strong expression and overproduction of OprF. Furthermore outer membrane porin was examined by MALDI-TOF MS/MS with a Mascot Score of 222 against bacteria database.

Created the MtrCAB BioBrick <bbpart>K1172401</bbpart>
Engineered MtrCAB devices with different promotors and RBS: (<bbpart>BBa_K1172403</bbpart>, <bbpart>BBa_K1172404</bbpart>, <bbpart>BBa_K1172405</bbpart>)

We supported the CeBiTec student academy with supervising the ‘Synthetic Biology’ experiment and the day of synthetic biology in the city of Bielefeld.
Our team assigned the Track Food & Energy in this year, with the project title ‘Ecolectricity – currently available’.

We successfully produced the alanine racemase biobrick <bbpart>BBa_K1172901</bbpart> also with ptac promoter (<bbpart>BBa_K1172902</bbpart>), with double terminator (<bbpart>BBa_K1172903</bbpart>) and the Barnase biobrick <bbpart>BBa_K1172904</bbpart>.
We successfully produced our biosafety systems araCtive (<bbpart>BBa_K1172909</bbpart>), TetOR alive (<bbpart>BBa_K1172915</bbpart>) and lac of growth (<bbpart>BBa_K1172908</bbpart>).

Riboflavin synthesis gene-cluster brought to BioBrick form <bbpart>BBa_K1172303</bbpart> and equipped with three different promoters, resulting in three new devices:

<bbpart>BBa_K608002</bbpart> + <bbpart>BBa_K1172303</bbpart> = <bbpart>BBa_K1172306</bbpart>
<bbpart>BBa_K608006</bbpart> + <bbpart>BBa_K1172303</bbpart> = <bbpart>BBa_K1172305</bbpart>
<bbpart>BBa_K525998</bbpart> + <bbpart>BBa_K1172303</bbpart> = <bbpart>BBa_K1172304</bbpart>

Characterization of <bbpart>BBa_K1172303</bbpart> starts with absorbance measurement of produced riboflavin.
Potential riboflavin supplementary genes norM and ribC converted to BioBrick standard:

Successful cultivations of Escherichia coli KRX with OprF and GldA in our Microbial Fuel Cell.
- NADH overproduction for the GldA strain improves extracellular electron transfer mediated by NADH and resulting in an 20 % increased bioelectricity output.
- Escherichia coli KRX with OprF shows 100 % higher voltage and electric charge than E. coli wild type. Electron shuttle-mediated electron transfer across the membrane is greatly improved by heterologous expression of outer membrane porin OprF.

Our preferred riboflavin production strain, E. coli KRX equipped with <bbpart>BBa_K1172306</bbpart> , was thouroughly characterized.

The overexpression of Riboflavin synthase beta subunit RibE, which belongs to the rib-gene-cluster <bbpart>BBa_K1172303</bbpart> . was examined by MALDI-TOF MS/MS with a Mascot Score of 906 against the NCBI database.
We proofed the production of riboflavin qualitatively through LC/MS measurement.
Measuring the fluorescence of supernatant samples from our riboflavin production strain together with riboflavin solutions of known concentration enabled us to make predications regarding the amount of riboflavin produced by our strain.
Measurement of supernatant samples from our riboflavin production strain against wild type samples with HPLC confirmed an approximately 60fold overexpression of riboflavin and backed up our result from fluorescence and absorbance measurements.

European Jamboree:
- Advance to Championship
- Best Presentation, Europe, Overgrad
- Regional Finalist, Europe, Overgrad
- Grand Prize Winner, Europe, Overgrad

We constructed new devices by combination of the mediator producing parts (riboflavin synthesis gene cluster and glycerol dehydrogenase) with the oprF coding sequence as well as oprF under control of the T7 promoter.