Difference between revisions of "Part:BBa K2260001"
 
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<font size="4"><b>phaC1-J4 with polyhistidine tags</b></font>
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<font size="4"><b>phaC1J4 with polyhistidine tags</b></font>
  
 
<h2> Overview</h2>
 
<h2> Overview</h2>
  
 
<p style="text-indent: 4em;">
 
<p style="text-indent: 4em;">
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In order to utilize short and medium chain length volatile fatty acids (VFAs) our part consists of phaC1 (<i>Pseudomonas aeruginosa</i>) and phaJ4 (<i>Pseudomonas putida</i>). Transcription of phaJ4 leads to expression of enoyl-coA hydratase and pha synthase from phaC1. These enzymes are involved in the pathway that leads to conversion of volatile fatty acids (VFAs) such as acetic acid, propionic acid, butyric acid, etc. to poly[(R)-3-hydroxybutyrate] (PHB). The gene construct also includes histidine tags upstream of each gene and contains two ribosome binding sites (RBS).
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This part consists of phaC1 (<i>Pseudomonas aeruginosa</i>) and phaJ4 (<i>Pseudomonas putida</i>), which allow <i>E. coli</i> to convert short- and medium-chain-length volatile fatty acids (VFAs) to PHB. The phaJ4 gene codes for enoyl-coA hydratase, while the phaC1 gene codes for pha synthase. The construct also contains the RBS <a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> upstream of each coding region and histidine tags at the N-termini of each protein.
 
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</p>
 
<p style="text-indent: 4em;">
 
<p style="text-indent: 4em;">
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This part was inserted into pET29(b)+ downstream a T7 promoter and lacZ. Thus, expression of phaC1J4 gene was induced using  Isopropyl β-D-1-thiogalactopyranoside (IPTG). <i>E. coli </i> (BL21) was transformed with the plasmid containing the gene construct. The bacteria was used in our experiments to test the ability of the part to synthesize PHB in different conditions. The three different conditions used were pure VFAs only, fermented synthetic feces supernatant (which is referred as "syn poo" supernatant on this page), and glucose only.
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This part was inserted into pET29(b)+ downstream a T7 promoter and lacZ repressor. Thus, expression of phaC1J4 gene was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). <i>E. coli </i> BL21(DE3) was transformed with the plasmid containing the gene construct. The bacteria was used in our experiments to test the ability of the part to synthesize PHB in different conditions. The three different conditions used were pure VFAs only, fermented synthetic feces supernatant, and glucose only.
 
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<h2> PHB from fermented "syn poo" supernatant</h2>
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<h2> PHB from Fermented Synthetic Feces Supernatant</h2>
 
<p style="text-indent: 4em;">
 
<p style="text-indent: 4em;">
 
In order to test the construct in the different conditions, overnights (O/Ns) of our construct and negative control (<i>E. coli</i> transformed with pET29(b)+ containing no insert) was were used. The O/Ns were grown for ~24 hours and the OD<sub>600</sub> was adjusted to be in the range 0.4-0.7. The table below shows the OD<sub>600</sub> readings taken before inoculation.
 
In order to test the construct in the different conditions, overnights (O/Ns) of our construct and negative control (<i>E. coli</i> transformed with pET29(b)+ containing no insert) was were used. The O/Ns were grown for ~24 hours and the OD<sub>600</sub> was adjusted to be in the range 0.4-0.7. The table below shows the OD<sub>600</sub> readings taken before inoculation.
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<p> Three replicates were carried out for each of the conditions. Our construct and negative control O/Ns were added to the media containing carbon sources. The composition of the media in each of the twelve 125 mL Erlenmeyer flasks is given below:
 
<p> Three replicates were carried out for each of the conditions. Our construct and negative control O/Ns were added to the media containing carbon sources. The composition of the media in each of the twelve 125 mL Erlenmeyer flasks is given below:
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<center><p style="text-indent: 2em;"> <b>Table 2.</b> Negative control and our construct in three different conditions: glucose only, pure VFAs only, and "syn poo" supernatant.</p></center>
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<center><p style="text-indent: 2em;"> <b>Table 2.</b> Negative control and our construct in three different conditions: glucose only, pure VFAs only, and fermented synthetic feces supernatant.</p></center>
 
<center><img width="600" height="350" src="https://static.igem.org/mediawiki/2017/4/4e/Beta_exp_table.png"></center>
 
<center><img width="600" height="350" src="https://static.igem.org/mediawiki/2017/4/4e/Beta_exp_table.png"></center>
  
 
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The cells were allowed to grow in the media for ~24 hours and centrifuged. Cells were then resuspended in (1x) PBS for extraction and the OD<sub>600</sub> was recorded before proceeding to other steps for extraction. The table below shows the recorded OD<sub>600</sub>.
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The cells were allowed to grow in the media for ~24 hours and centrifuged. Cells were then resuspended in 1X PBS for extraction and the OD<sub>600</sub> was recorded before proceeding to other steps for extraction. The table below shows the recorded OD<sub>600</sub>.
 
</p>
 
</p>
 
<center><p style="text-indent: 2em;"> <b>Table 3.</b> The OD<sub>600</sub> readings of cells resuspended in (1x) PBS. </p></center>
 
<center><p style="text-indent: 2em;"> <b>Table 3.</b> The OD<sub>600</sub> readings of cells resuspended in (1x) PBS. </p></center>
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<center><p style="text-indent: 2em;"> <b>Figure 1.</b> Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on far right and other tubes containing our construct in the different conditions.</p></center>
 
<center><p style="text-indent: 2em;"> <b>Figure 1.</b> Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on far right and other tubes containing our construct in the different conditions.</p></center>
  
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<h2> HPLC of PHB</h2>
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<h2>HPLC of PHB Digested in Sulphuric Acid</h2>
 
<p style="text-indent: 4em;">
 
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Karr <i>et. al</i> showed that digestion of PHB in sulphuric acid leads to the production of crotonic acid (1983). The protocol used for PHB digestion is given <a href="http://2017.igem.org/Team:Calgary/Experiments">here</a>. About 0.025 g of PHB was digested for 15 mins and 30 mins. For each of the durations low and high dilution factors were used. Figure 2, 3, 4 and 5 show the HPLC results.
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Karr <i>et. al</i> (1983) showed that digestion of PHB in sulphuric acid leads to the production of crotonic acid. The protocol used for PHB digestion is given <a href="http://2017.igem.org/Team:Calgary/Experiments">here</a>. About 0.025 g of PHB was digested for 15 mins and 30 mins. For each of the durations low and high dilution factors were used. Figure 2, 3, 4 and 5 show the HPLC results.
 
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<h2> References</h2>
 
<h2> References</h2>
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<p  style="text-indent: 2em;">Karr DB, Waters JK, Emerich DW. Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection . Applied and Environmental Microbiology. 1983;46(6):1339-1344.</p>
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<p  style="text-indent: 2em;">Karr DB, Waters JK, Emerich DW. (1983). Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection . Applied and Environmental Microbiology. 1983;46(6):1339-1344.</p>
 
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Latest revision as of 03:49, 2 November 2017

phaC1J4 with polyhistidine tags

Overview

This part consists of phaC1 (Pseudomonas aeruginosa) and phaJ4 (Pseudomonas putida), which allow E. coli to convert short- and medium-chain-length volatile fatty acids (VFAs) to PHB. The phaJ4 gene codes for enoyl-coA hydratase, while the phaC1 gene codes for pha synthase. The construct also contains the RBS BBa_B0034 upstream of each coding region and histidine tags at the N-termini of each protein.

This part was inserted into pET29(b)+ downstream a T7 promoter and lacZ repressor. Thus, expression of phaC1J4 gene was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). E. coli BL21(DE3) was transformed with the plasmid containing the gene construct. The bacteria was used in our experiments to test the ability of the part to synthesize PHB in different conditions. The three different conditions used were pure VFAs only, fermented synthetic feces supernatant, and glucose only.

PHB from Fermented Synthetic Feces Supernatant

In order to test the construct in the different conditions, overnights (O/Ns) of our construct and negative control (E. coli transformed with pET29(b)+ containing no insert) was were used. The O/Ns were grown for ~24 hours and the OD600 was adjusted to be in the range 0.4-0.7. The table below shows the OD600 readings taken before inoculation.

Table 1. The O/Ns were grown for ~24 hours and OD600 was adjusted and recorded in the table.

Three replicates were carried out for each of the conditions. Our construct and negative control O/Ns were added to the media containing carbon sources. The composition of the media in each of the twelve 125 mL Erlenmeyer flasks is given below:

Table 2. Negative control and our construct in three different conditions: glucose only, pure VFAs only, and fermented synthetic feces supernatant.

The cells were allowed to grow in the media for ~24 hours and centrifuged. Cells were then resuspended in 1X PBS for extraction and the OD600 was recorded before proceeding to other steps for extraction. The table below shows the recorded OD600.

Table 3. The OD600 readings of cells resuspended in (1x) PBS.

After extraction of PHB from cells using sodium hypochlorite extraction method the final weights of tube containing the product was weighed and recorded as follows:

Table 4. Initial weight of 50 mL Falcon tubes was recorded. Final weight of tube + PHB extracted was recorded. Finally, final weight - initial weight was used to calculate the amount of PHB extracted from the cells in 50 mL cultures.

Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on far right and other tubes containing our construct in the different conditions.

HPLC of PHB Digested in Sulphuric Acid

Karr et. al (1983) showed that digestion of PHB in sulphuric acid leads to the production of crotonic acid. The protocol used for PHB digestion is given here. About 0.025 g of PHB was digested for 15 mins and 30 mins. For each of the durations low and high dilution factors were used. Figure 2, 3, 4 and 5 show the HPLC results.

Low digestion time, low dilution factor

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 15 mins and dilution factor of 15.

Low digestion time, high dilution factor

Figure 3. HPLC results from digestion of PHB in sulphuric acid for 15 mins and dilution factor of 30 (replicate 2).

High digestion time, low dilution factor

Figure 4. HPLC results from digestion of PHB in sulphuric acid for 30 mins and dilution factor of 15.

High digestion time, high dilution factor

Figure 5. HPLC results from digestion of PHB in sulphuric acid for 30 mins and dilution factor of 30.

Discussion of HPLC results

The HPLC results had a crotonic acid peak for all samples. This confirmed that the obtained product was PHB. Although we were successful at detecting PHB using HPLC, the obtained amount of crotonic acid may be lower than the actual amount produced as the method of quantification of PHB using HPLC requires further development. In particular, an optimal digestion time needs to be selected so that all of the PHB in the sample digests to crotonic acid without further degradation of crotonic acid. The PHB extraction method can also be optimized to minimize the amount of PHB lost during extraction.

References

Karr DB, Waters JK, Emerich DW. (1983). Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection . Applied and Environmental Microbiology. 1983;46(6):1339-1344.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]