Difference between revisions of "Team:Amsterdam/Project/Eng rom/Photosyn car"

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Acetate production involved the knock-out of the native acs gene and heterologous over-expression of the ackA1 and pta genes.</p>
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Acetate production involved the knock-out of the native acs gene and heterologous over-expression of the ackA1 gene from <i>Lactocuccus lactis</i> and pta gene from <i>Synechocystis</i>.</p>
  
 
  <figure class ="image fit">
 
  <figure class ="image fit">
 
   <img src="https://static.igem.org/mediawiki/2015/a/a0/Acetate_pathway.png" alt=" Acetate pathway">
 
   <img src="https://static.igem.org/mediawiki/2015/a/a0/Acetate_pathway.png" alt=" Acetate pathway">
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   <figcaption style="color: #2C3539">Figure 3. - The acetate pathway we targeted. The acs gene represents the recycling reaction that is knocked-out to enable growth-coupled production, while ackA1 and pta were targeted to further increase acetate production. </figcaption>
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   <figcaption style="color: #2C3539">Figure 4. - The acetate pathway we targeted. The acs gene represents the recycling reaction that is knocked-out to enable growth-coupled production, while ackA1 and pta were targeted to further increase acetate production. </figcaption>
 
</figure>  
 
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<p> The following constructs were used for the knocking out of acs and insertion of ackA1, pta, or fused AckA1/pta:
 
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</p>
 
 
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<div class="6u">
 
<div class="6u">
  
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<p>
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<p> The acs gene codes for acetyl-CoA synthase, an enzyme that converts acetate into acetyl CoA. This is the acetate recycling reaction in the biomass formation pathway. The reactions that actually lead to the formation of acetate as a side-product represent a two-step process and are governed by pta, coding for the phosphate acetyl-transferase enzyme that converts acetyl-CoA into acetyl-phosphate, and ack, coding for an acetate kinase that removes acetyl-phosphate's phosphate group to produce acetate (technically functioning as a phosphatase). Although over-expressing ackA1 and pta via gene insertions is prone to fixating mutations, we still wanted to see its impact on overall acetate production. The following constructs were used for the knocking out of acs and insertion of ackA1, pta, or fused AckA1/pta:
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This system allow us to reduce the time of observing a mutation but now it comes the second problem. How do we know if a mutation leading to a change in the parameters has happened? The first option that comes to our synthetic biologist mind is by sequencing periodically the genome of the population and screen it for mutations. The major drawback of sequencing is that it does not inform whether the mutation affects the parameters or not. The remaining solution is then to estimate the physiological parameters with high time definition to spot when the mutation has occurred. Using the turbidostat it would be possible to store all the values recorded by the spectrophotometer and calculate the growth rate from these data. This is done by fitting a linear model to the logarithm of the OD over the time. The production rate can obtained by periodically measuring the amount of product in the medium (link to protocols for enzymatic assays and HPLC).
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  <figure class ="image fit">
 
  <figure class ="image fit">
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   <img src="https://static.igem.org/mediawiki/2015/b/b6/Amsterdam_MC.png" alt=" Turbidostat">
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   <img src="https://static.igem.org/mediawiki/2015/d/dd/Screen_Shot_2015-09-18_at_19.35.20.png" alt="acetate constructs">
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   <figcaption style="color: #2C3539">Figure 4. - The multicultivator. </figcaption>
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   <figcaption style="color: #2C3539">Figure 5. - Constructs used to knock-out acs and over-express ackA1 and pta. All plasmids contained homologous sequences of neutral sites in the Synechocystis genome that enabled homologous recombination to take place in order to insert or eliminate target genes. AckA1 and pta were expressed via the constitutive pcpc promoter. </figcaption>
 
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Luckily for us  when we came to the lab there was a master student, Joeri Jongbloets{link}, who had transformed a multicultivator (MC) like this{link} in a 8 channel turbidostat. For his internship he wrote the program that connect with the MC hardware and control the pumps to dilute the culture when necessary. In addition the software stores the measurements from the MC spectrophotometers in a database and analyzes it to obtain the growth rate. Thanks to this impressive piece of software engineering we were able to run long term experiments and observe evolution.  
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After the genetic engineering steps, batch and turbidostat cultivation experiments were conducted to measure acetate production and stability. Acetate concentrations were measured using the Megazyme acetate assay kit.
 
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<h4>Stability</h4>
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<h4>Stable acetate production</h4>
 
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<p> In Our turbidostat experiments we observed the results we expected. In the lactate strain (figure 1), a non growth coupled carbon producer, we started to observe an increase in growth rate about 300 hours after the beginning of the experiment. At the same time point we recorded a drop in the production of lactate. This result confirms our hypothesis about how mutations leading to the loss of production can be quickly fixated inasmuch as it releases the burden imposed by the carbon production increasing the fitness of the mutated cells.  
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<p> By knocking out the acs gene, we expected to eliminate the acetate recycling reaction in the biomass formation pathway, which would lead to acetate accumulation in growth-coupled fashion. Although this is what the model indicated should happen, modelling results hardly ever translate perfectly to real-world scenarios. In order to test the Synechocystis acs mutant's acetate production (only acs was knocked out for this strain, ack and pta were not yet overexpressed), we conducted the turbidostat experiments described in more detail on this <a href = "https://2015.igem.org/Team:Amsterdam/Project/Phy_param/Synechocysytis">page</a>.
 
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<figure class ="image fit">
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  <img src="https://static.igem.org/mediawiki/2015/6/68/Amsterdam_qp_acetate.png" alt="acetate constructs">
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  <figcaption style="color: #2C3539">Figure 6. - Turbidostat results showing stable production of acetate by a Synechocystis acs mutant over long periods of time.</figcaption>
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On the other hand the growth coupled acetate producer, &Delta;acs, shown a constant production and growth rate over the length of the experiment (figure 2) demonstrating that <a href = "https://2015.igem.org/Team:Amsterdam/Project/Eng_rom/Photosyn_car">this strategy</a> is more stable than the classical engineering approach. The  Q<sub>p</sub> estimated around 400 hours shows a decrease in the production but it is probably due to experimental failure. In the turbidostat cells were grown at very low OD (threshold was set to 0.35) therefore the concentration of acetate was below the detection limit of the enzymatic assay method. To overcome this problem we dehydrate the samples by lyophilization, also called freeze-drying, then dilute them in an smaller volume therefore increasing the concentration above the detection limit. Although this method worked (we obtained similar Q<sub>p</sub> values using different culture systems), the sample processing could have introduce noise in this measurement.  
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We indeed observed stable acetate production over time, maintained well over 900 hours until the end of the experiment. The contrast this result offers to the lactate results shows that knocking out the acetate recycling reaction in the biomass-formation pathway represents a succesful strategy for inducing stable production.
 
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<h4>Parameters</h4>
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<h4>Ramping up production</h4>
 
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In table 2 the physiological parameters obtained for six strains producing glycerol, lactate and acetate are presented. The lactate strain shown the highest Q<sub>p</sub> followed by &Delta;acs and Syn1413. An intriguing results is the differences in Q<sub>p</sub> and growth rate of &Delta;acs between cultivation methods. In the turbidostat the length of the exponential phase from where the growth rate is calculated is smaller than in photostat which could be influencing the estimation. For the Q<sub>p</sub> the previously discussed argument could also be an explanation for this difference. In addition it is possible that the photostat conditions are more favourable for the growth of <i>Synechocystis</i>.  
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In an attempt to further increase acetate production and increase the production capacity of our consortium, we took the acs mutant described above and over-expressed the ackA1 gene and pta gene - encoding the two reactions that lead to acetate formation in the biomass formation pathway. During the engineering of the pta strain, we discovered several mutations had occurred in pta fragment, resulting in a frame-shift mutation that removed the stop codon and extended the amino acid chain. We therefore created a second strain from scratch, but included the first strain to investigate the impact of the frameshift mutations. The three resulting strains, ackA1, pta mutated, and pta, were cultivated in a photostat, a batch reactor with OD-dependent light intensity, over a period of four days with samples taken every four hours until an OD treshold was reached. The results shown in figure 6 and 7 show that although overall acetate concentrations were higher in the pta strain (again, this also has the acs KO background) compared to the acs mutant strain, the average Q<sub>p</sub>'s are higher for the acs mutant. These results suggest that over-expressing ackA1 or pta does not result in increased acetate production any more than simply knocking out the acs gene does.
 
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   <img src="https://static.igem.org/mediawiki/2015/f/f8/Amsterdam_table_qp.png" alt="Acetate Qp and growth">
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   <img src="https://static.igem.org/mediawiki/2015/e/ef/Amsterdam_table_qp_dav.png
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   <figcaption>Table 1. - Estimated Growth and Q<sub>p</sub> for different <i>Synechocystis</i> strains in the two cultivation systems.</figcaption>
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" alt="Acetate Qp and growth">
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   <figcaption>Table 1. - Estimated Growth and Q<sub>p</sub> for ackA1 and pta over-expressing acs mutant <i>Synechocystis</i> strains in the two cultivation systems.</figcaption>
 
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Revision as of 19:47, 18 September 2015

iGEM Amsterdam 2015

Engineering Stable Carbon Production

Sharing is Carbon

Overview

Background

The driving force of our consortium's romance is the phototrophic carbon-sharing module: an engineered Synechocystis that fixates CO2 and produces compounds that can be used by a chemotroph to create desired end-products like biofuels.

Aim

Productive relationships need to stand the test of time. Having experienced the perils of instability firsthand, we sought to engineer carbon production in way that would last.

Approach

Using genetic engineering strategies guided by modelling results, we used a combination of gene knock-outs and over-expressions to target a pathway that would result in growth-coupled acetate production.

Results

  • We engineered growth-coupled acetate production by knocking out the acs gene, showing that this indeed leads to stable acetate production
  • We over-expressed the Pta and AckA1 genes to increase the flux towards acetate formation.
.

Connections

Acetate Qp and growth
Figure 1. - Growth and Qp of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.
Qp and growth
Figure 2. - Acetate pathway of Synechocystis, showing how acetate is produced and recycled in the biomass-formation pathway.

Change of plans

Different carbon compounds produced by Synechocystis could serve as fuel for E. coli in our consortium: Glucose, lactate, and glycerol for example, are all products that an engineered cyanobacteria can produce.

Turbidostat
Figure 3. - Potential carbon compounds that Synechocystis can produce.

Although glucose seems like a great compound to drive sustainable bio-production with, our initial plan was to develop Synechocystis strains that produce all of these products, and to compare their performances in a consortium with E. coli generating different products. After the initial results of lactase production came in, however, we decided to make genetic stability the central focus of our carbon fixation efforts: engineering a Synechocystis that produces a carbon compound in the most stable way possible such that its relationships with other consortia-species last more than one or a few nights. Based on the results of our software tools, which parsed the genome-scale metabolic map to search for compounds that could be produced in a growth-coupled way, we decided to engineer acetate production.

Methods

As the genetic engineering of carbon production in Synechocystis involved relatively simple constructs, standard restriction-digestion cloning protocols were used for most engineering efforts. Synechocystis knock-outs were created using the specific markerless knock-out method described by [ref]. This two-round knock-out approach allowed for the cultivation of knock-out strains without the need of antibiotics and thus increased flexibility of implementation in a potential consortium.

Acetate production involved the knock-out of the native acs gene and heterologous over-expression of the ackA1 gene from Lactocuccus lactis and pta gene from Synechocystis.

Acetate pathway
Figure 4. - The acetate pathway we targeted. The acs gene represents the recycling reaction that is knocked-out to enable growth-coupled production, while ackA1 and pta were targeted to further increase acetate production.

The acs gene codes for acetyl-CoA synthase, an enzyme that converts acetate into acetyl CoA. This is the acetate recycling reaction in the biomass formation pathway. The reactions that actually lead to the formation of acetate as a side-product represent a two-step process and are governed by pta, coding for the phosphate acetyl-transferase enzyme that converts acetyl-CoA into acetyl-phosphate, and ack, coding for an acetate kinase that removes acetyl-phosphate's phosphate group to produce acetate (technically functioning as a phosphatase). Although over-expressing ackA1 and pta via gene insertions is prone to fixating mutations, we still wanted to see its impact on overall acetate production. The following constructs were used for the knocking out of acs and insertion of ackA1, pta, or fused AckA1/pta:

acetate constructs
Figure 5. - Constructs used to knock-out acs and over-express ackA1 and pta. All plasmids contained homologous sequences of neutral sites in the Synechocystis genome that enabled homologous recombination to take place in order to insert or eliminate target genes. AckA1 and pta were expressed via the constitutive pcpc promoter.

After the genetic engineering steps, batch and turbidostat cultivation experiments were conducted to measure acetate production and stability. Acetate concentrations were measured using the Megazyme acetate assay kit.

Results

Stable acetate production

By knocking out the acs gene, we expected to eliminate the acetate recycling reaction in the biomass formation pathway, which would lead to acetate accumulation in growth-coupled fashion. Although this is what the model indicated should happen, modelling results hardly ever translate perfectly to real-world scenarios. In order to test the Synechocystis acs mutant's acetate production (only acs was knocked out for this strain, ack and pta were not yet overexpressed), we conducted the turbidostat experiments described in more detail on this page.

acetate constructs
Figure 6. - Turbidostat results showing stable production of acetate by a Synechocystis acs mutant over long periods of time.

We indeed observed stable acetate production over time, maintained well over 900 hours until the end of the experiment. The contrast this result offers to the lactate results shows that knocking out the acetate recycling reaction in the biomass-formation pathway represents a succesful strategy for inducing stable production.

Ramping up production

In an attempt to further increase acetate production and increase the production capacity of our consortium, we took the acs mutant described above and over-expressed the ackA1 gene and pta gene - encoding the two reactions that lead to acetate formation in the biomass formation pathway. During the engineering of the pta strain, we discovered several mutations had occurred in pta fragment, resulting in a frame-shift mutation that removed the stop codon and extended the amino acid chain. We therefore created a second strain from scratch, but included the first strain to investigate the impact of the frameshift mutations. The three resulting strains, ackA1, pta mutated, and pta, were cultivated in a photostat, a batch reactor with OD-dependent light intensity, over a period of four days with samples taken every four hours until an OD treshold was reached. The results shown in figure 6 and 7 show that although overall acetate concentrations were higher in the pta strain (again, this also has the acs KO background) compared to the acs mutant strain, the average Qp's are higher for the acs mutant. These results suggest that over-expressing ackA1 or pta does not result in increased acetate production any more than simply knocking out the acs gene does.

Lactate Qp and growth
Figure 1. - Growth rate (median value in segments of 48 hours with a band of 1 standard deviation) and Qp (dots represent technical replicates) of strain SAA023 over 900 hours of continuous culture showing the drop in production.

Acetate Qp and growth
Figure 2. - Growth rate (median value in segments of 48 hours with a band of 1 standard deviation) and Qp (dots represent technical replicates)of strain Δacs over 900 hours of continuous culture showing a constant production over the duration of the experiment.

Acetate Qp and growth
Table 1. - Estimated Growth and Qp for ackA1 and pta over-expressing acs mutant Synechocystis strains in the two cultivation systems.