Difference between revisions of "Part:BBa K1172304"
 
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<partinfo>BBa_K1172304 short</partinfo>
 
<partinfo>BBa_K1172304 short</partinfo>
  
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This gene cluster consists of four different genes that form a single operon.
 
  
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[[Image:IGEM_Bielefeld_Riboflavin_6WellPlate.jpg|500px|thumb|center|<p align="justify"> '''Figure 1: From left to right: ''e. coli'' KRX wild type compared to ''e. coli'' KRX with <bbpart>BBa_K1172303</bbpart> under control of a medium constitutive promoter (<bbpart>BBa_K525998</bbpart>) and ''e. coli'' KRX with <bbpart>BBa_K1172303</bbpart> under control of a strong constitutive promoter (<bbpart>BBa_K608002</bbpart>).]]</p>
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<p align="justify">
 +
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.
 +
</p>
 
===Usage and Biology===
 
===Usage and Biology===
 
<p align="justify">
 
<p align="justify">
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[http://de.wikipedia.org/wiki/Riboflavin Riboflavin], or Vitamin B2 is a redox-active substance that plays an
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essential role in   living cells. Secreted into the medium, it can be
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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.
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effectively used by some bacteria for electron transfer. Presence of riboflavin in anaerobic cultures leads to higher current flow in a
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</p>
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microbial fuel cell, which made riboflavin overproduction a suitable target for optimisation of our MFC.
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[[File:IGEM Bielefeld 2013 riboflavin.jpg|200px|thumb|left|<p align="justify">'''Figure 2: '''Riboflavin (Vitamin B2) and flavin-coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide).]]</p>
 +
 
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<p align="justify">
 +
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.
 +
</p>
 +
 
 +
<p align="justify">
 +
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.
 
<br>
 
<br>
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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
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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.
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significant riboflavin overproduction detectable both in supernatant and in cells.
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</p>
 
</p>
  
 
<!-- -->
 
<!-- -->
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<br><br><br><br><br><br>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1172304 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1172304 SequenceAndFeatures</partinfo>
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===Results===
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===Part uses===
  
 +
For overproduction of riboflavin, the BioBrick <bbpart>BBa_K1172303</bbpart> was combined with promoters of different strenghts.
 +
{| class="wikitable" style="margin-left:0px;"
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!Device: <bbpart>BBa_K1172303</bbpart>
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under control of accordant promoter.
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!Promoters used
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!RBS / Activity
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|-
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|<bbpart>BBa_K1172304</bbpart>
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|Anderson 0.33 -> |<bbpart>BBa_K525998</bbpart> “T7 induced"
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|[https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 strong] / very strong
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|-
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|<bbpart>BBa_K1172305</bbpart>
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|<bbpart>BBa_K608006</bbpart> “Anderson 0.33”
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|[https://parts.igem.org/wiki/index.php?title=Part:BBa_B0032 medium] / medium
 +
|-
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|<bbpart>BBa_K1172306</bbpart>
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|<bbpart>BBa_K608002</bbpart> “Anderson 0.77”
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|[https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 strong] / strong
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|}
  
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==Results==
 
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===Confirming overexpression of the rib-gene cluster===
 
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The overexpression of <bbpart>BBa_K1172303</bbpart> and its derived devices <bbpart>BBa_K1172306</bbpart>,<bbpart>BBa_K1172305</bbpart>, <bbpart>BBa_K1172304</bbpart> is assured by verifying the protein Riboflavin synthase beta subunit RibE
 
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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.
 
  
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====SDS-PAGE====
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<!-- -->
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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.
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[[File:IGEM-Bielefeld-2013-Rib-SDS-20-perc.jpg|450px|thumb|center|<p align="justify"> '''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 '''</p>]]
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====MALDI-TOF====
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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.
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[[Image:iGEM_Bielefeld_2013_Maldiergebnis_screenshot_2.10.13_Week22.jpg|400px|thumb|left|<p align="justify"> '''Figure 11: Exported MALDI-TOF results. '''</p>]][[Image:iGEM_Bielefeld_2013_MALDIergebnis_2.10.13_Week22.jpg|400px|thumb|center|<p align="justify"> '''Figure 12:
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Screenshot of the BioTools user interface showing the pure results of the MALDI-TOF. '''</p>]]
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===Analysis of riboflavin in supernatants===
 
  
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====Absorbance measurement====
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Since the Regional in Lyon, we were able to combine <bbpart>BBa_K1172303</bbpart> with other BioBricks from our project.
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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 <bbpart>BBa_K1172306</bbpart> 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. <br>
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[[Image:iGEM_Bielefeld_2013_absorbancetable_4.10.13.jpg|450px|thumb|center|<p align="justify"> '''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 <bbpart>BBa_K1172306</bbpart>, sn = supernatant, cd = cell disruption.'''</p>]] <br>
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{| class="wikitable" style="margin-left:0px;"
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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 <bbpart>BBa_K1172306</bbpart> and  6112.63 µg /L for the cell disruption samples of ''E. coli''  KRX with <bbpart>BBa_K1172306</bbpart>. The concentration of putative riboflavin in the wild type strain was not detectable.
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!Device: Combination of <bbpart>BBa_K1172303</bbpart>
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:* Absorbance measurement is the least sensitive method used for riboflavin detection. Therefore the slightly higher yields should be taken with a grain of salt.
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with accordant parts
 +
!Part 1 of the new device
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!Explanation
 +
|-
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|<bbpart>BBa_K1172588</bbpart>
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| |<bbpart>BBa_K1172502</bbpart>
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|Combination of ''oprF'' from ''P. fluorescence'' under control of T7 promoter and the ''rib''-gene-cluster from ''S. oneidensis''
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|-
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|<bbpart>BBa_K1172599</bbpart>
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|<bbpart>BBa_K1172501</bbpart>  
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| Combination of the coding sequences ''oprF'' from ''P. fluorescence'' and the ''rib''-gene-cluster from ''S. oneidensis''
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|}
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 +
 
 +
<!-- -->
 +
 
 +
 
 +
===Results===
  
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====Fluorescence measurement====
 
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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 <bbpart>BBa_K1172306</bbpart>  (grown for 72 hours) , ''E. coli'' KRX with <bbpart>BBa_K1172306</bbpart>  (grown for 12 hours) and ''E. coli'' KRX wild type bacteria (grown for 72 hours) <br>
 
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[[Image:iGEM_Bielefeld_2013_fluoreszenzmessung_table2_4.10.13.jpg|450px|thumb|center|<p align="justify"> '''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 <bbpart>BBa_K1172306</bbpart>, sn = supernatant, cd = cell disruption.'''</p>]] <br>
 
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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.
 
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The concentration of putative riboflavin in the wild type strain was not detectable.
 
  
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====HPLC measurement====
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This part was used to characterize the riboflavin synthesis gene cluster from ''shewanella oneidensis'' (<bbpart>BBa_K1172303</bbpart>). Please look at the Parts Registry page for <bbpart>BBa_K1172303</bbpart> for results and detailed information.  
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Supernatant and cell disruption samples of ''E. coli'' KRX with BBa_K1172306  (grown for 72 hours) , ''E. coli'' KRX with <bbpart>BBa_K1172306</bbpart> (grown for 12 hours) and ''E. coli''  KRX wild type bacteria (grown for 72 hours) were measured in a HPLC detector.
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[[Image:iGEM_Bielefeld_2013_ribos_hplc_resulttable_4.10.13.jpg|400px|thumb|left|<p align="justify"> '''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. '''</p>]][[Image:iGEM_Bielefeld_2013_ribos_hplc_zentriert_4.10.13.jpg|400px|thumb|center|<p align="justify"> '''Figure 13: Results of the HPLC measurement shown as graph. Figure 13 was centered on the riboflavin peak for a better view. '''</p>]]
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Latest revision as of 15:29, 28 October 2013

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


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
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1152
  • 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.