Difference between revisions of "Part:BBa K2260000"
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<center><img width="600" height="420" src="https://static.igem.org/mediawiki/2017/e/e7/CBA_experiment_table.png"></center> | <center><img width="600" height="420" src="https://static.igem.org/mediawiki/2017/e/e7/CBA_experiment_table.png"></center> | ||
| − | <center><p style="text-indent: 2em;"> Table 1. Recorded OD<sub>600</sub> of O/Ns before inoculating the media containing VFAs, OD<sub>600</sub> of cells after growing in media for ~24 hours, then centrifuged and resuspended in (1x) PBS for extraction. Initial weight of 50 ml falcon tubes and final weights of tube containing PHB in grams was recorded. </p></center> | + | <center><p style="text-indent: 2em;"> <b>Table 1.</b> Recorded OD<sub>600</sub> of O/Ns before inoculating the media containing VFAs, OD<sub>600</sub> of cells after growing in media for ~24 hours, then centrifuged and resuspended in (1x) PBS for extraction. Initial weight of 50 ml falcon tubes and final weights of tube containing PHB in grams was recorded. </p></center> |
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<center><img width="600" height="420" src="https://static.igem.org/mediawiki/2017/c/c5/CBA_plastic.png"></center> | <center><img width="600" height="420" src="https://static.igem.org/mediawiki/2017/c/c5/CBA_plastic.png"></center> | ||
| − | <center><p style="text-indent: 2em;"> Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on left and phaCBA on right.</p></center> | + | <center><p style="text-indent: 2em;"> <b>Figure 1.</b> Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on left and phaCBA on right.</p></center> |
<h2>HPLC of PHB</h2> | <h2>HPLC of PHB</h2> | ||
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<center><h4 >Low</h4></center> | <center><h4 >Low</h4></center> | ||
<center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/b/b3/HPLC_CBA_Low.png"></center> | <center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/b/b3/HPLC_CBA_Low.png"></center> | ||
| − | <center><p style="text-indent: 2em;"> Figure 2. HPLC results from digestion of PHB in sulphuric acid for 20 mins.</p></center> | + | <center><p style="text-indent: 2em;"><b> Figure 2. </b>HPLC results from digestion of PHB in sulphuric acid for 20 mins.</p></center> |
<center><h4 >High</h4></center> | <center><h4 >High</h4></center> | ||
<center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/6/6a/HPLC_CBA_High.png"></center> | <center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/6/6a/HPLC_CBA_High.png"></center> | ||
| − | <center><p style="text-indent: 2em;"> Figure 3. HPLC results from digestion of PHB in sulphuric acid for 30 mins.</p></center> | + | <center><p style="text-indent: 2em;"> <b>Figure 3. </b>HPLC results from digestion of PHB in sulphuric acid for 30 mins.</p></center> |
<center><h4 >Arbitrary</h4></center> | <center><h4 >Arbitrary</h4></center> | ||
<center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/6/6e/HPLC_CBA_Arbitrary.png"></center> | <center><img style="vertical-align: bottom;" width="620" height="520" src="https://static.igem.org/mediawiki/2017/6/6e/HPLC_CBA_Arbitrary.png"></center> | ||
| − | <center><p style="text-indent: 2em;"> Figure 4. HPLC results from digestion of arbitrary amount of PHB in sulphuric acid for 30 mins.</p></center> | + | <center><p style="text-indent: 2em;"> <b>Figure 4.</b> HPLC results from digestion of arbitrary amount of PHB in sulphuric acid for 30 mins.</p></center> |
Revision as of 04:51, 30 October 2017
Overview
The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes acetyl-coA, which is a product of the glycolysis pathway ___!!source!!____. Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA ___!!source!!____.
In order to utilize acetic acid present in fermented human feces ___!!source!!____, we decided to incorporate the phaCAB operon. However, literature has shown that the rearrangement of operon to phaCBA leads to higher amount of production of PHB ___!!source!!____. Thus, we obtained the operon from BBa_K1149051 and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.
PHB Weights from fermented "syn poo" supernatant
Our part was tested for production of PHB using PHB synthesis using VFAs as feedstock protocol. There were 9 replicates for our part in the "syn poo" supernatant and 3 replicates of the negative control. The OD600 of overnights (O/Ns) was measures before inoculating the media containing VFAs. PHB was extracted using sodium hypochlorite extraction method. After resuspension of cells in 1x PBS for extraction the OD600 was recorded to estimate the number of cells. The following table summarizes the data:
Table 1. Recorded OD600 of O/Ns before inoculating the media containing VFAs, OD600 of cells after growing in media for ~24 hours, then centrifuged and resuspended in (1x) PBS for extraction. Initial weight of 50 ml falcon tubes and final weights of tube containing PHB in grams was recorded.
Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on left and phaCBA on right.
HPLC of PHB
It is known from literature that digestion of PHB in sulphuric acid leads to production of crotonic acid. We analyzed PHB obtained by digesting PHB using the PHB digestion in sulphuric acid protocol. About 0.581 g of PHB was obtained and digested for 20 mins (Low) and 30 mins (High). Lastly, an arbitrary amount was digested for 30 mins. The following figures shows the HPLC results obtained from the Low, High, and Arbitrary samples:
Low
Figure 2. HPLC results from digestion of PHB in sulphuric acid for 20 mins.
High
Figure 3. HPLC results from digestion of PHB in sulphuric acid for 30 mins.
Arbitrary
Figure 4. HPLC results from digestion of arbitrary amount of PHB in sulphuric acid for 30 mins.
The HPLC results showed that a peak for crotonic acid was seen. This confirmed the white powder obtained was PHB. However, the area of crotonic acid was low due to a number of limitations.
Nile red suspension for confirmation of PHB
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]