Growth Assays

Media characterisation

All medias tested

LB

M9 Minimal

M9 Supplemented

Waste Conditioned Media

Waste Assessment

(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.

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 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.

Product inhibition

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