Revision as of 00:51, 10 December 2019

M E A S U R E M E N T

Metabolic Engeneering

One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds with CO₂ and light as a resource. As a target, we thought about molecules, which also tackle one of the most important topic: the climate change.

Planes are considered to be one of the most environmentally damaging transport devices commonly used (Borken-Kleefeld et al., 2010). A quick estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO₂ was released from the flights. To encounter this problem, we chose to set our focus on farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50% limonene, 40% farnesene) from Amyris (Brennan et al., 2015). This fuel has proven to be a suitable alternative to chemically produced, oil based jet fuels.

Metabolic MEP Pathway — Fig. 1- Overview of the MEP-Pathway. Enzymes are marked blue and Green. Limonene and farnesene Synthase are summarised as TPS. Modified after Lin et al 2016

Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene synthase. Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (Lin et al., 2016).

As a the first step we decided to heterologous express the limonene and the farnesene synthase to establish overexpression strains. We used the limonene synthase from Lavandula angustifolia and the farnesene synthase from Actinidia deliciosa and codon optimised both enzymes. To redirect the flux into the MEP-Pathway we decided to overexpress the E.Coli proteins DXS (1-deoxy-D-xylulose-5-phosphate synthase), IDI (Isopentenyl-diphosphate Delta-isomerase) and IspA (Farnesyl diphosphate synthase). These targets were chosen on based previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the growth rate (Choi et al.,2016). Surprisingly, this suggest strong capacities of production in Cyanobacteria and no build-up of toxic intermediates.

Many pathways, such as the MEP-Pathway, are regulated on the first committing step. Therefore we chose the DXS as a first target. The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour of IPP is required.

IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a farnesene production strain. To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a phenylalanine (F). This would lead to a threefold increase in activity in vitro (Xiang et al., 2016). Also it would be advisable to remove the allosteric feedback regulation of the DXS (Banerjee et al.,2016).

Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extracellular diffusion rate is enough to prevent intracellular and potential toxic accumulation. On the other hand, a nontoxic overlay to catch the molecules is required, for example dodecan can be used. (Lee et al., 2017). An alternative supply with CO2 is also required, therefore we copied the system from Lee et al. and introduced a small tube with holes into the Medium.

To balance the pathway we decided to use a weak promotor (BBa_J23103) for the ispA, as competition over FPP could lead to a heavy growth impact (Lin et al., 2016). As IDI is an Isomerase, only a comparably medium amount of protein should be sufficient for a enhanced effect, so Promotor BBa_J23110 was chosen. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor BBa_J23111.

Sadly, we weren´t able to test our constructs in vivo and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( BBa_K3228051 and BBa_K3228052).

Storytelling:

We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in front of many problems and questions. Especially the usage of different media, light conditions and other cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic microbiology and necessary to compare results with other scientists and reproduce their data.

asas — Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra.

While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in light measurement, evaluated different reporters???, established a measurement method and compared it to a already known FACS measurement method (?).

At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. So we had to measure the light conditions in our incubators and while doing this simple task the first part of standardization began. We discovered that nearly every paper? is using different methods to measure their light conditions and that it is a really complex and important procedure. So we got in contact with cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show different ways of measurement, their (dis-)advantages and different results depending on the measuring instrument.
Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In literature and while talking with different experts (IHP), we recognized that small deviations of these parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters that lead to the fastest growth speed.
Another aspect was measuring the expression and characterize our part. Different possibilities were discussed and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.
The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the difference in measurement methods.
At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 and make a contribution to the cyano community by establishing essential/fixed standards in measurement.

L I G H T
M E A S U R E M E N T

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R E P O R T E R S

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F A C S

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P A R T
M E A S U R E M E N T

For our project it was indispensable to establish a measurement workflow that is not only applicable to UTEX 2973 and other cyanobacteria but also has a high throughput.

For our project it was indispensable to establish a measurement workflow that is not only applicable to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement method that suites such a large collection. Therefore we elaborated different workflows - containing different cultivation vessels and parameters - and revised them after evaluating the results. In the end we were able to establish a workflow specially designed for our methods to cultivate and characterize the parts from our Marburg Collection 2.0, that is tailored to Synechococcus elongatus UTEX 2973.

Experimental Procedure

The results of our part characterization were obtained by fluorescence and luminescence measurements (of what?). But before the party could be measured we had to elaborate a cultivating and measuring workflow.
For the cultivating workflow we tested different well plate formats and growing parameters for the best growing conditions. It was logistically the best way to cultivate and measure the parts in well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space in our incubator. Starting with 96-well-plates it was impossible to cultivate Synechococcus elongatus UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of the incubator was limited whereas cultures in flasks had to be incubated at the same time and these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating flasks and well-plates in the same incubator. After revising the workflow over and over we came to the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. Further it was necessary to use transparent wells to ensure every well with similar ight conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an important role in cultivation of well plates cause the realtive small volumes and high surfaces (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is essential to know the volume in the wells for measuring in the plate reader. Therefore we compared different seals for the well plates and in the end we came to the conclusion that using a semipermeable foil is the best solution. The evaporation could be minimalized and the cells were able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to cultivate the cells for 2-3 days without losing significant amounts of medium.

xxxx Fig x.:Schema vom Workflow
As described before we used the following workflow as shown in fig. XX to cultivate and measure our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the end of the triparental conjugation (LINK). For every part we picked 3 different colonies and inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to OD₇₃₀=0.6-0.8 they were inoculated to 1.0 mL of OD₇₃₀=0.1 into the wells A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was inoculated with 1.0 mL of a OD₇₃₀= 0.1 UDAR culture that was used as a blank while evaluating the results (that will be used as a blank while ...). When all the cultures in the second 24-well-plate reached OD₇₃₀=0.6-0.8 they got inoculated twice in the same well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When the wells C1-D6 (and the UDAR) reached an OD₇₃₀=0.6-0.8 the cultures were transferred into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three times. Following this workflow we were able to measure three biological parallels and two technical parallels for every biological parallel. It enabled us to have a good statistical database and gives our results a stronger meaning/significance. While working with this workflow it was essential to keep the cultures in their exponential phase because it would significantly speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es erst gar keine lag phase gibt).
Concerning the measurement part we decided to transfer the cultures into black/white luminescence is measured in white ones. We measured in 96-well-plates because it enabled us to measure every part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for measurement)

Fluorescence measurement:
After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the considered part. For measurement we created a program that measured the OD₇₃₀ and the fluorescence of the wells.

fig XX (screenshot des messprogams)
In order to measure the OD in each well we determined the absorption at 730 nm. Further we measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the border of the well showed consistent results with small standard deviations (fig. XX). We used the same settings of the multiple measurement for the fluorescence measurement. While using sYFP as signal for our part measurement we have set the emission wavelength to 515 nm and the excitation wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database verlinken/als quelle?)

Fluorescence-Activated Cell Sorting (FACS):
short abstract and link to the FACS-text of the measurement

Luminescence Measurement

text

Data analysis and evaluation

kein plan was man hier schreiben soll zum jetzigen standpunkt... For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
Auswertung, Daten und Grafen darstellen?

G R O W T H
C U R V E S

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Abstract?

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Unterprojekt2

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One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds with CO₂ and light as a resource. As a target, we thought about molecules, which also tackle one of the most important topic: the climate change.

Planes are considered to be one of the most environmentally damaging transport devices commonly used (Borken-Kleefeld et al., 2010). A quick estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO₂ was released from the flights. To encounter this problem, we chose to set our focus on farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50% limonene, 40% farnesene) from Amyris (Brennan et al., 2015). This fuel has proven to be a suitable alternative to chemically produced, oil based jet fuels

Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene synthase. Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (Lin et al., 2016).

As a the first step we decided to heterologous express the limonene and the farnesene synthase to establish overexpression strains. We used the limonene synthase from Lavandula angustifolia and the Farnesen synthase from Actinidia deliciosa and codon optimised both enzymes. To redirect the flux into the MEP-Pathway we decided to overexpress the E.Coli proteins DXS, IDI and IspA. These targets were chosen on based previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the growth rate (Choi et al.,2016). Surprisingly, this suggest strong capacities of production in Cyanobacteria.

We chose the DXS, as many pathways are regulated on the first committing step. The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, which is especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour of IPP is required.

IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a farnesene production strain. To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a phenylalanine (F). This would lead to a threefold increase in activity in vitro (Xiang et al., 2016). Also it would be advisable to remove the allosteric feedback regulation of the DXS (Banerjee et al.,2016).

Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extracellular diffusion rate is enough to prevent intracellular and potential toxic accumulation. On the other hand, a nontoxic overlay to catch the molecules is required, for example dodecan can be used. (Lee et al., 2017). An alternative supply with CO2 is also required, therefore we copied the system from Lee et al. and introduced a small tube with holes into the Medium.

To balance the pathway we decided to use a weak promotor (BBa_J23103) for the ispA, as competition over FPP could lead to a heavy growth impact (Lin et al., 2016). As IDI is an Isomerase, only a comparably small amount of protein should be sufficient for a enhanced effect, so Promotor BBa_J23110 was chosen. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor BBa_J23111.

Sadly, we weren´t able to test our constructs in vivo and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( K3228051 and K3228052).

@@ Line 143: / Line 143: @@
 </p>
 <p>
-Sadly, we weren´t able to test our constructs <i>in vivo</i> and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> K3228051</a> and <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> K3228052</a>).
+Sadly, we weren´t able to test our constructs <i>in vivo</i> and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> BBa_K3228051</a> and <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> BBa_K3228052</a>).
 </p>

Difference between revisions of "Team:Marburg/test"

Revision as of 00:51, 10 December 2019

M E A S U R E M E N T

Metabolic Engeneering

Storytelling:

L I G H T M E A S U R E M E N T

Light Measurement

R E P O R T E R S

Reporter

Unterprojekt1

Unterprojekt2

F A C S

Fluorescence-Activated Cell Sorting (FACS)

Unterprojekt1

Unterprojekt2

P A R T M E A S U R E M E N T

Part Measurement

Experimental Procedure

Data analysis and evaluation

G R O W T H C U R V E S

Growth Curves

Unterprojekt1

Unterprojekt2

L I G H T
M E A S U R E M E N T

P A R T
M E A S U R E M E N T

G R O W T H
C U R V E S