Part:BBa_K1172306

Riboflavin synthesis gene cluster from s. oneidensis under control of a strong Anderson promoter

Figure 1: From left to right: e. coli KRX wild type compared to e. coli KRX with BBa_K1172303 under control of a medium constitutive promoter (BBa_K525998) and e. coli KRX with BBa_K1172303 under control of a strong constitutive promoter (BBa_K608002).

This gene cluster consists of four different genes that form a single operon. These genes are pivotal in the riboflavin biosythesis pathway of Shewanella oneidensis and are transcribed polycystronic. The original operon in Shewanella oneidensis has additional activator and repressor genes. It was observed that these are not sufficient for riboflavin overproduction. Therefore these genes were not isolated from genomic DNA.

Usage and Biology

Riboflavin, or Vitamin B2 is a redox-active substance that plays an essential role in living cells. As precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) it is crucial for diverse energy supplying metabolic processes, e.g. beta-oxidation or oxidative phosphorylation. Riboflavin is water soluble and shows a distinct yellow coloration. For that reason it is also used for food coloration. It is easily detectable through absorbance and fluorescence measurement. Due to its fluorescent properties and non-toxicity it is used to detect leaks or to control cleaning processes.

Figure 2: Riboflavin (Vitamin B2) and flavin-coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide).

Riboflavin consists of two functional subunits, a short-chain ribitol and a tricyclic heterosubstituted isoalloxazine ring. The latter, also known as a riboflavin ring, exists in three redox states and is responsible for the diverse chemical activities of riboflavin. A fully oxidized quinone, a one-electron semiquinone and a fully reduced hydroquinone state are the three stages of riboflavin oxidation. In an aqueous solution, the quinone (fully oxidized) form of riboflavin has a typical yellow coloring. It becomes red in a semi-reduced anionic or blue in a neutral form and is colorless when fully reduced.

Secreted into the medium, it can be effectively used by some bacteria for electron transfer. Presence of riboflavin in anaerobic cultures leads to higher current flow in a Microbial Fuel Cell, which makes riboflavin overproduction a suitable target for optimisation of our MFC.
We have shown that cloning of the riboflavin cluster from a metal-reducing bacterium Shewanella oneidensis MR-1 in E. coli is sufficient to achieve significant riboflavin overproduction detectable both in supernatant and in cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1175
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Part uses

For overproduction of riboflavin, the BioBrick BBa_K1172303 was combined with promoters of different strenghts.

Device: BBa_K1172303 under control of accordant promoter.	Promoters used	RBS / Activity
BBa_K1172304	BBa_K525998 “T7 induced"	strong / very strong
BBa_K1172305	BBa_K608006 “Anderson 0.33”	medium / medium
BBa_K1172306	BBa_K608002 “Anderson 0.77”	strong / strong

Since the Regional in Lyon, we were able to combine BBa_K1172303 with other BioBricks from our project.

Device: Combination of BBa_K1172303 with accordant parts	Part 1 of the new device	Explanation
BBa_K1172588	BBa_K1172502	Combination of oprF from P. fluorescence under control of T7 promoter and the rib-gene-cluster from S. oneidensis
BBa_K1172599	BBa_K1172501	Combination of the coding sequences oprF from P. fluorescence and the rib-gene-cluster from S. oneidensis

Results

This part was used to characterize the riboflavin synthesis gene cluster from shewanella oneidensis (BBa_K1172303). Please look at the Parts Registry page for BBa_K1172303 for results and detailed information.