Team:Imperial College/data
From 2013.igem.org
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<h1>Media characterisation</h1> | <h1>Media characterisation</h1> | ||
- | < | + | <h4 class="clear">Growth assays with different experimental media</h4> |
[[File:LB_M9.png|thumbnail|left|500px|<b>Figure 1: The medias used for our growth assays using the MG1655 strain.</b> phaCAB is an operon involved in synthesis of P3HB while EV represents an empty vector. SR refers to stress response cells containing a red pigment. M9M is minimal media, while M9S is supplemented minimal media, as described [https://2013.igem.org/Team:Imperial_College/Protocols#M9_minimal_and_supplemented_media here]. WCM refers to waste conditioned media, which is made from sterile filtrated SRF waste, see [https://2013.igem.org/Team:Imperial_College/Protocols#Waste_Conditioned_Media_.28WCM.29 here]. Error bars are SEM, n=4.]] | [[File:LB_M9.png|thumbnail|left|500px|<b>Figure 1: The medias used for our growth assays using the MG1655 strain.</b> phaCAB is an operon involved in synthesis of P3HB while EV represents an empty vector. SR refers to stress response cells containing a red pigment. M9M is minimal media, while M9S is supplemented minimal media, as described [https://2013.igem.org/Team:Imperial_College/Protocols#M9_minimal_and_supplemented_media here]. WCM refers to waste conditioned media, which is made from sterile filtrated SRF waste, see [https://2013.igem.org/Team:Imperial_College/Protocols#Waste_Conditioned_Media_.28WCM.29 here]. Error bars are SEM, n=4.]] | ||
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- | < | + | <h3>Waste media </h3> |
We can see from this experiment whether our bacteria can grow solely on the mixed waste. | We can see from this experiment whether our bacteria can grow solely on the mixed waste. | ||
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- | < | + | <h3>Waste conditioned media</h3> |
{| class="wikitable" style="margin: 1em auto 1em auto;" | {| class="wikitable" style="margin: 1em auto 1em auto;" | ||
|[[File:WCM_media.png|thumbnail|right|400px|Growth curve of our [http://parts.igem.org/Part:BBa_K639003 mCherry] MG1655 bacteria. MG1655 were grown with LB media and sterile filtrated WCM at 37ºC. Error bars represents SE of the mean, n=4]] | |[[File:WCM_media.png|thumbnail|right|400px|Growth curve of our [http://parts.igem.org/Part:BBa_K639003 mCherry] MG1655 bacteria. MG1655 were grown with LB media and sterile filtrated WCM at 37ºC. Error bars represents SE of the mean, n=4]] | ||
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- | < | + | <h3>Empty Vector Control</h3> |
[[File:EV.png]] | [[File:EV.png]] | ||
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<h1>Characterisation of existing Biobricks</h1> | <h1>Characterisation of existing Biobricks</h1> | ||
- | < | + | <h3>phaCAB biobrick characterisation</h3> |
- | < | + | <h4 class="clear">LB</h4> |
[[File:LB.png|thumbnail|center|500px|<b>Figure 1: MG1655 in LB with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. LB shows the strongest growth curve with minimal latency. Error bars are SEM, n=4.]] | [[File:LB.png|thumbnail|center|500px|<b>Figure 1: MG1655 in LB with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. LB shows the strongest growth curve with minimal latency. Error bars are SEM, n=4.]] | ||
- | < | + | <h4 class="clear">M9 Minimal</h4> |
[[File:M9M.png|thumbnail|center|500px|<b>Figure 1: MG1655 in M9M with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. M9M shows the least growth growth of all the medias as it has low carbon and amino acid content. Error bars are SEM, n=4.]] | [[File:M9M.png|thumbnail|center|500px|<b>Figure 1: MG1655 in M9M with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. M9M shows the least growth growth of all the medias as it has low carbon and amino acid content. Error bars are SEM, n=4.]] | ||
- | < | + | <h4 class="clear">M9 Supplemented</h4> |
[[File:M9S.png|thumbnail|center|500px|<b>Figure 1: MG1655 in M9S with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. M9S shows a lag phase in growth but quickly increases due to increased amino acid content, nearly reaching LB after 5h. Error bars are SEM, n=4.]] | [[File:M9S.png|thumbnail|center|500px|<b>Figure 1: MG1655 in M9S with plasmids EV and phaCAB.</b> There is no growth inhibition when comparing the empty vector with the phaCAB vector in each media. M9S shows a lag phase in growth but quickly increases due to increased amino acid content, nearly reaching LB after 5h. Error bars are SEM, n=4.]] | ||
- | < | + | <h3>Stress biosensor characterisation BBa_K639003</h3> |
- | < | + | <h4>IPTG induction assay</h4> |
Originally we intended on using [http://parts.igem.org/Part:BBa_K639003 BBa_K639003] to detect whether our cells were stressed when placed in various toxic byproducts. However, as the data below shows, this biobrick is very leaky. As such, we are using the stress sensor as a marker for cell growth and also to show that the cells had been successfully transformed with the correct chloramphenicol resistance. | Originally we intended on using [http://parts.igem.org/Part:BBa_K639003 BBa_K639003] to detect whether our cells were stressed when placed in various toxic byproducts. However, as the data below shows, this biobrick is very leaky. As such, we are using the stress sensor as a marker for cell growth and also to show that the cells had been successfully transformed with the correct chloramphenicol resistance. | ||
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- | < | + | <h3>pBAD characterisation</h3> |
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- | < | + | <h3>Glucose </h3> |
[[File:Glucose_graph.png|thumbnail|center|600px|Cell growth of phaABC <i>E. coli</i> at 4 concentrations of glucose. Optimum growth is at 2-4% glucose at 37ºC. Error bars represents SE of the mean, n=4]] | [[File:Glucose_graph.png|thumbnail|center|600px|Cell growth of phaABC <i>E. coli</i> at 4 concentrations of glucose. Optimum growth is at 2-4% glucose at 37ºC. Error bars represents SE of the mean, n=4]] | ||
<br> | <br> | ||
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<br> | <br> | ||
- | < | + | <h3>Plastic Toxicity Assays</h3> |
- | < | + | <h4>L-lactic Acid </h4> |
[[File:L-LActic_Acid.png|thumbnail|center|600px|Cell growth of MG1655 on 5mM L-Lactic Acid. Error bars represents SE of the mean, n=4.]] | [[File:L-LActic_Acid.png|thumbnail|center|600px|Cell growth of MG1655 on 5mM L-Lactic Acid. Error bars represents SE of the mean, n=4.]] | ||
<h3>Ethylene glycol </h3> | <h3>Ethylene glycol </h3> | ||
[[File:EG_growth.png|thumbnail|center|600px|Cell growth of MG1655 in ethylene glycol, a byproduct of polyurethane degradation. Cells were grown in 0mM, 100mM or 200mM Ethylene Glycol at 30ºC. Error bars represents SE of the mean, n=4]] | [[File:EG_growth.png|thumbnail|center|600px|Cell growth of MG1655 in ethylene glycol, a byproduct of polyurethane degradation. Cells were grown in 0mM, 100mM or 200mM Ethylene Glycol at 30ºC. Error bars represents SE of the mean, n=4]] | ||
- | < | + | <h4>3-hydroxybutyrate (3HB) </h4> |
[[File:3HB666.png]] | [[File:3HB666.png]] | ||
<h3>Acetoacetate </h3> | <h3>Acetoacetate </h3> | ||
[[File:AA777.png]] | [[File:AA777.png]] | ||
- | < | + | <h4>Poly(3-hydroxybutyrate) P(3HB) </h4> |
[[File:31ug_P3HB.png]] | [[File:31ug_P3HB.png]] | ||
- | < | + | <h4>Poly(lactic acid) (PLA) </h4> |
[[File:PLA.png]] | [[File:PLA.png]] | ||
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- | < | + | <h3>Sole carbon source</h3> |
- | < | + | <h4>3HB</h4> |
[[File:3HB_sole_carbon_source.png]] | [[File:3HB_sole_carbon_source.png]] | ||
- | < | + | <h4>Acetoacetate</h4> |
[[File:AA_sole_carbon_source.png]] | [[File:AA_sole_carbon_source.png]] | ||
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<h1>PHB production</h1> | <h1>PHB production</h1> | ||
- | < | + | <h3>Nile red staining</h3> |
O/N cutures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining. The staining indicates the production of P3HB. | O/N cutures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining. The staining indicates the production of P3HB. | ||
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- | < | + | <h3>Purification of P3HB</h3> |
|[[File:IMG_2191.JPG|thumbnail|left|200px|P(3HB) purified from phaCAB transformed MG1655 that were grown in LB with 3% glucose]] | |[[File:IMG_2191.JPG|thumbnail|left|200px|P(3HB) purified from phaCAB transformed MG1655 that were grown in LB with 3% glucose]] |
Revision as of 21:07, 23 September 2013
Contents
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Media characterisation
Growth assays with different experimental media
Waste Assays
These assays were designed to test whether our chassis could grow either directly with waste and/or in waste conditioned media. Waste conditioned media is a filter sterilised version of the waste media and was designed for several reasons; we were unsure whether mixed waste would be toxic to Ecoli and hence a less concentrated version may be more suitable and secondly large chunks of waste would prevent accurate OD600 measurements and therefore we decided to filter out the largest chunks.
Waste media
We can see from this experiment whether our bacteria can grow solely on the mixed waste.
Conclusion MG1655 can survive solely on mixed waste.
Waste conditioned media
Characterisation of our Biobricks
Empty Vector Control
Characterisation of existing Biobricks
phaCAB biobrick characterisation
LB
M9 Minimal
M9 Supplemented
Stress biosensor characterisation BBa_K639003
IPTG induction assay
Originally we intended on using [http://parts.igem.org/Part:BBa_K639003 BBa_K639003] to detect whether our cells were stressed when placed in various toxic byproducts. However, as the data below shows, this biobrick is very leaky. As such, we are using the stress sensor as a marker for cell growth and also to show that the cells had been successfully transformed with the correct chloramphenicol resistance.
pBAD characterisation
Glucose
ANOVA analysis shows that...
Plastic Toxicity Assays
L-lactic Acid
Ethylene glycol
3-hydroxybutyrate (3HB)
Acetoacetate
Poly(3-hydroxybutyrate) P(3HB)
Poly(lactic acid) (PLA)
Sole carbon source
3HB
Acetoacetate
Western blots
Enzyme Kinetics
PHB production
Nile red staining
O/N cutures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining. The staining indicates the production of P3HB.
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Purification of P3HB
| | |