Difference between revisions of "Part:BBa K1172304"

Revision as of 03:51, 5 October 2013

Riboflavin synthesis gene cluster from s. oneidensis under control of T7 promoter and strong RBS

This gene cluster consists of four different genes that form a single operon.

Usage and Biology

[http://de.wikipedia.org/wiki/Riboflavin Riboflavin], or Vitamin B2 is a redox-active substance that plays an essential role in living cells. 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 made 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

Results

Results

Confirming overexpression of the rib-gene cluster

The overexpression of BBa_K1172303 and its derived devices BBa_K1172306,BBa_K1172305, BBa_K1172304 is assured by verifying the protein Riboflavin synthase beta subunit RibE The protein RibE is part of the riboflavin synthesis pathway of Shewanella oneidensis. The corresponding gene is ribE. RibE belongs to the rib-gene cluster, which we managed to isolate, removing all the illegal restriction sites and subsequently cloned into pSB1C3.

SDS-PAGE

The performed SDS-PAGE shows a distinct band at ~15 kDa. The exact size of the riboflavin synthase beta subunit RibE is 16.7 kDa. The band was cut out and analyzed by MALDI-TOF.

Figure 10: SDS-PAGE with 20% separating gel for the verification of proteins from the rib-cluster. From left to right: Thermo PageRuler 150 kDa prestained ladder; E. coli KRX wild type 1; E.coli KRX wild type 2; rib-T7 uninduced; rib-T7 induced; rib-medium-Anderson33; rib-strong-Anderson77

MALDI-TOF

The spot, described above, was picked and digested with trypsine. Afterwards the sample was spotted on the target and analyzed by MALDI-TOF Measurement of the sample produced valid data: RibE was examined by MALDI-TOF MS/MS with a Mascot Score of 906 against the NCBI database concerning bacterial organisms.

Figure 11: Exported MALDI-TOF results.

Figure 12: Screenshot of the BioTools user interface showing the pure results of the MALDI-TOF.

Analysis of riboflavin in supernatants

Absorbance measurement

Riboflavin has an absorption peak at 446 nm. The absorbance was measured in a TECAN infinite plate reader. The samples consisted of supernatant derived from E. coli KRX with BBa_K1172306 and KRX as the "wild type" (both strains were cultivated over 72 hours). Further intracellular measurements of both strains were obtained. Therefore, the cells were disrupted via a ribolisation step, centrifugated and the yielded supernatend was evaluated.

Table 1: Pipetting scheme and measurement results of riboflavin standards and cell samples for absorbance measurement at 446 nm in the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]. WT = wild type, And77 = Coli equipped with BBa_K1172306, sn = supernatant, cd = cell disruption.

Riboflavin in known concentrations (5.31 * 10^-5 M) and dilutions was measured to generate a calibration curve. The subsequently computed riboflavin concentrations were 5773.3 µg / L for the supernatant of E. coli KRX with BBa_K1172306 and 6112.63 µg /L for the cell disruption samples of E. coli KRX with BBa_K1172306. The concentration of putative riboflavin in the wild type strain was not detectable.

• Absorbance measurement is the least sensitive method used for riboflavin detection. Therefore the slightly higher yields should be taken with a grain of salt.

Fluorescence measurement

Riboflavin absorbs light at 440 nm with a corresponding emission at 535 nm. The fluorescence was measured in a TECAN infinite plate reader. The samples consisted of supernatant samples from E. coli KRX with BBa_K1172306 (grown for 72 hours) , E. coli KRX with BBa_K1172306 (grown for 12 hours) and E. coli KRX wild type bacteria (grown for 72 hours)

Table 2: Pipetting scheme and measurement results of riboflavin standards and cell samples for fluorescence measurement, emission at 535 nm. Measured in the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]. WT = wild type, And77 = Coli equipped with BBa_K1172306, sn = supernatant, cd = cell disruption.

Riboflavin in known concentrations and dilutions was measured to generate a calibration line. The subsequently computed riboflavin concentrations were 308.1 µg / L for the supernatant sample after 12 hours and 3821.5 µg /L for the supernatant sample after 72 hours. The concentration of putative riboflavin in the wild type strain was not detectable.

HPLC measurement

Supernatant and cell disruption samples of E. coli KRX with BBa_K1172306 (grown for 72 hours) , E. coli KRX with BBa_K1172306 (grown for 12 hours) and E. coli KRX wild type bacteria (grown for 72 hours) were measured in a HPLC detector.

Table 3: HPLC measurement results for riboflavin concentrations in supernatant (sn) and cell disruption (cd) samples after 72 hours and 12 hours of cultivation respectively.

Figure 13: Results of the HPLC measurement shown as graph. Figure 13 was centered on the riboflavin peak for a better view.