Media characterisation

Growth assays with different experimental media

Figure 1: The medias used for our growth assays using the MG1655 strain. 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 here. WCM refers to waste conditioned media, which is made from sterile filtrated SRF waste, see here. Error bars are SEM, n=4.

Waste and waste conditioned media growth assays

(A) WCM precursor material, this sterilised media made from LB and SRF was used to produce all WCM utilised. (B) Cells containing mCherry pigment grown in SRF (A) over 3 days, then streaked in a qualitative assay to check for growth. (C) mCherry cells were streaked again after 7 days growth in SRF.

(A) SRF in PBS (phosphate buffered saline), a buffer. We can see from this experiment whether our bacteria can grow solely on the waste SRF. (B) Cells containing mCherry pigment grown in SRF (A) over 3 days, then streaked in a qualitative assay to check for growth. (C) mCherry cells were streaked again after 6 days growth in SRF.

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

Production of the red pigment by stress induction. MG1655 were grown with LB media and sterile filtrated WCM for 48 hours.

Biobrick characterisation

phaCAB biobrick characterisation

LB

Figure 1: MG1655 in LB with plasmids EV and phaCAB. 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.

M9 Minimal

Figure 1: MG1655 in M9M with plasmids EV and phaCAB. 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.

M9 Supplemented

Figure 1: MG1655 in M9S with plasmids EV and phaCAB. 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.

PUR enzyme characterisation

Stress biosensor characterisation

Waste Conditioned Media

Figure 1: MG1655 in WCM with plasmids EV and SR. There is no growth inhibition when comparing the empty vector with the SR vector in each media. WCM contains LB but trends in a simliar manner to M9S, most likely due to toxins present within it from the SRF. Error bars are SEM, n=4.

pBAD characterisation

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Glucose

Cell growth of phaABC E. coli at 4 concentrations of glucose. Optimum growth is at 2-4% glucose at 37ºC. Error bars represents SE of the mean, n=4

Cell growth over 6h in LB and M9M minimal media. LB grown MG1655 phaCAB grow more rapidly initially then M9M but reach the same OD after 6h while EV shows a different trend. EV in M9M levels off at a much lower OD at 4h, as seen with EV grown in LB. Error bars are SEM, n=4.

Cell growth over 6h in LB and M9S minimal media. LB grown MG1655 phaCAB grow more rapidly initially then M9S but after 5h, phaCAB in M9S continue to grow to a higher OD. EV shows a different trend, in M9S it levels off at a similar level to LB. Error bars are SEM, n=4.

ANOVA analysis shows that...

Plastic Toxicity Assays

L-lactic Acid

Cell growth of MG1655 on 5mM L-Lactic Acid. Error bars represents SE of the mean, n=4.

Ethylene glycol

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

3-hydroxybutyrate (3HB)

Acetoacetate

Poly(3-hydroxybutyrate) P(3HB)

Poly(lactic acid) (PLA)

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.

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Sole carbon source

3HB

Acetoacetate

Induction

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Cell growth over 6h with IPTG induction. mCherry production is induced by the stress pathway and detection of ppGpps. In order to bypass this, we induced with IPTG which inhibits LacI, resulting in mCherry expression.

Fluorescence of the cells under IPTG induction over a 6h period.

Empty Vector Control

Western blots

Enzyme Kinetics

PHB production