Team:TU Darmstadt/Results/Pathway
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- | <!--TYPO3SEARCH_begin--><div id="c234" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Overview</h1></div><p>We | + | <!--TYPO3SEARCH_begin--><div id="c234" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Overview</h1></div><p>We build up a pathway consisting of seven enzymes (TAL, 4CL, CHS, CHI, F3H, DFR, ANS), which we split into two operons. In addition we build up a biosensor for naringenin, which alowed us to prove this importnant intermediate. |
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<p>Now, we are satiesfied to present 3 results. | <p>Now, we are satiesfied to present 3 results. |
Revision as of 16:56, 17 October 2014
Overview
We build up a pathway consisting of seven enzymes (TAL, 4CL, CHS, CHI, F3H, DFR, ANS), which we split into two operons. In addition we build up a biosensor for naringenin, which alowed us to prove this importnant intermediate.
Now, we are satiesfied to present 3 results.
1. Our naringenin operon (K1497016) functions well.
2. Our naringenin biosensor (K1497019 - K1497022) works in several constructions.
3. Our pelargonidin operon (K1497015) functions well and we could extract the desired anthocyanin.
Naringenin operon (K1497016)
This part is a composite of four genes each with the strong RBS (BBa_B0034).
- 4-Coumaryl ligase - 4CL (BBa_K1033001)
- Tyrosine ammonia lyase - TAL (BBa_K1033000)
- Chalchone isomerase - CHI (BBa_K1497100)
- Chalchone synthase - CHS (BBa_K1497101)
Together, these genes build the naringenin biosynthesis operon without a promotor. In addition of a promotor part the device is able to build S-naringenin. These device is working in E. coli K and B strains.
![](fileadmin/_processed_/csm_Naringenin_Operon-_genetic_map_cf99d65d46.png)
We created the naringenin biosynthesis operon under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . We measured the naringenin production after a 16 h incubation time with the naringenin biosensor BBa_K1497020.
The cell pellets from E. coli BL21(DE3) - pSB1C3-fdeR-gfp with and without T7-naringenin operon (BBa_K1497017) are shown in figure 3. Only in the cell pellet with BBa_K1497017 exhibited GFP fluorescence.
We were also able to measure the GFP fluorescence quantitatively and to calculate with the help of a calibration curve for the naringenin sensor the production yield of both operons (Figure 4). For BBa_K1497017 we calculated 3 µM naringenin and for the operon with the constitutive promoter BBa_J23100 (BBa_K1497016) we calculated 1.9 µM naringenin.
![](fileadmin/_processed_/csm_naringeninoperont7coli_88646f3d62.png)
![](fileadmin/_processed_/csm_naringenBALK_7215128235.png)
fdeR - Naringenin sensor (K1497019 - K1497022)
FdeR is a homo dimeric protein from Herbaspirillum seropedicae. In the presence of naringenin (or naringenin chalchone), FdeR activates the specific promoter region upstream of the fdeR region and induces a strong gene expression. In Herbaspirillum seropedicae the FdeR activates the Fde-Operon (Fde: Flavanone degradation) and enable growth with naringenin and the naringenin chalcone.
So in combination with GFP or another fluorescense protein this part can be used as an in vivo naringenin sensor.
![](fileadmin/_processed_/csm_naringeninsensensorschemagruen_769e741a3b.png)
Usage and Biology
You can use the reporters for measuring naringenin concentrations in your samples.
Depending on which fluorophor you want to detect, you can use one of three biosensors:
![](fileadmin/_processed_/csm_petridischnaringenin_7dcbab788d.png)
You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use the Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.
Functional Parameter
The Biobrick BBa_K1497019 produces in E. coli B and K strains the FdeR Protein. We measured the fluorescense of GFP and mKate after the incubation with different concentrations of naringenin. The results are shown in Figure 7 & 8.
![](fileadmin/files/naringfp.png)
![](fileadmin/files/narinmrfp.png)
Pelargonidin operon (K1497015)
Pelargonidin is an anthocyanin. Anthocyanins are water-soluble vacuolar pigments that appear yellow to dark-blue (pH-dependent), which are responsible for color of flowers and fruits and are health-promoting for humans.
We constructed a pelargonidin producing operon under the control of a T7 promoter (K1497015). The operon consists of 3 genes (flavonon-3beta-hydroxylase, dihydroflavonol 4-reductase, anthocyanindin synthase) each with strong RBS (Fig.1) This operon catalyses the reaction from naringenin to pelargonidin (Fig. 2).
The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided from Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E. coli coding optimized and synthezised by Eurofins MWG opreon.
Futhermore, we designed two different ANS gene version by using XX to verify XXX
![](fileadmin/_processed_/csm_Genetic_map_ee08c72f6a.png)
![](fileadmin/_processed_/csm_PW2_c0efc4b41a.png)
Production
To analyze the pelagonidin production operon (K1497015), we transformed it into E.coli Bl21 (DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan et al., (2007). After the induction with 1mM Isopropyl-?-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9 media and fermented for 48h at 37°C in present of 0.1mM naringenin.
![](fileadmin/files/Results_PW1/Pelletfaerbung.png)
Extraction
After the expression of pelagonidin producing operon with engineered ANS (K1497015) in present of 0.5mM narigenin we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet and supernatant and verified the pH-dependency of pelargonidin (Fig. 3).
![](fileadmin/_processed_/csm_Methanol_ex_044d897bc8.png)
![](fileadmin/files/Results_PW1/DCM_ex.png)
Application
Construction of Grätzel cells
We used the extracted Pelargonidin (produced from E.coli Bl21 (DE3) containing the pelagonidin producing operon, K1497015) to build a Grätzel cell. This was done according to Michael Grätzel & Brian O'Reagan. We were able to produce voltage of 380 mV and 0.5 mA under daylight conditions.
![](fileadmin/_processed_/csm_graezel_cell_schema_0113082e76.png)
![](fileadmin/files/Graezelcell/eigene_Graetzel_cell_.png)