Team:Bilkent-UNAMBG/Results

Results

Bba_K1334002 (HxlR+P-formaldehyde) Induction

To design a formaldehyde sensor, initially Bba_K1334002 part, registered by iGEM14_WHU-China team in 2014, is tested. We tried induction with 0.5 mM and 1 mM formaldehyde and uninduced by using three biological replicas.

Figure 1 : Y axis corresponds to RFU (Relative Fluorescence Unit) 16 hours after formaldehyde addition and on the X axis sample groups are indicated. Voc: formaldehyde.

The overnight measurement indicates that the error bars are almost equal or more than the fold change of the each induction. This inconsistency can also be seen from the uninduced and 0.5 mM bars. Uninduced bar is almost 5 times more than the induced one. In conclusion, induction of the part Bba_K1334002 with formaldehyde is inconsistent which prompts us to improve this part.

HxlR/pFor Sensor Gradient aTc Induction

In order to control the expression level of HxlR protein, gradient aTc concentrations are utilized since HxlR gene expression is regulated by the promoter pL(tetO) within the HxlR/pFor sensor. Thus, HxlR protein is produced upon the addition of aTc and the amount of HxlR protein produced is positively correlated with the aTc concentration.

Figure 2 : Y axis corresponds to RFU (Relative Fluorescence Unit) 2 hours after formaldehyde addition and on the X axis sample groups are indicated. “(-)” denotes no addition of formaldehyde and “(+)” denotes 1 mM formaldehyde addition.

Figure 3 : Y axis corresponds to RFU (Relative Fluorescence Unit) 4 hours after formaldehyde addition and on the X axis sample groups are indicated. “(-)” denotes no addition of formaldehyde and “(+)” denotes 1 mM formaldehyde addition.

Figure 4 : Y axis corresponds to RFU (Relative Fluorescence Unit) 16 hours after formaldehyde addition and on the X axis sample groups are indicated. “(-)” denotes no addition of formaldehyde and “(+)” denotes 1 mM formaldehyde addition.

Gradient aTc induction results illustrated that elevated GFP signal upon the addition of formaldehyde is more significant when aTc concentration is 25 ng/μl.

Fluorescent and DIC Equipped Upright Microscope Results

After we did our induction, we looked our cells under Fluorescent and DIC Equipped Upright Microscope whether we can observe GFP signal color which is supposed to seen green according to signal rate:

Image 1. 40x zoom for 25ng/ul aTc concentration of HxlR construct after overnight measurement of induction.

Image 2. 40x zoom for 25ng/ul aTc and 1 mM VOC concentration of HxlR construct after overnight measurement of induction.

As it is seen from the images, comparison between them indicates that addition of VOC increases the green color density which refers that our formaldehyde biosensor is working.

HxlR/pFor Sensor Gradient Formaldehyde Induction

To determine the concentration of formaldehyde which would facilitate a higher fold change in GFP signal, gradient formaldehyde induction is performed. Since the most appropriate aTc concentration is determined in gradient aTc induction, all samples are initially induced with 25 ng/μl aTc. On the other hand, the least concentrated formaldehyde which provides a sufficient fold change, might be detected by gradient formaldehyde induction.

Figure 5 : Y axis corresponds to RFU (Relative Fluorescence Unit)16 hours after formaldehyde addition and on the X axis sample groups are indicated. Voc: formaldehyde.

The results of gradient formaldehyde induction depicted that 1 mM and 3.2 mM formaldehyde addition have a higher potency in GFP signal increase.

AlkR/pAlkM Sensor Gradient aTc Induction

In order to control the expression level of AlkR protein, gradient aTc concentrations are utilized since AlkR gene expression is regulated by the promoter pL(tetO) within the AlkR/pAlkM sensor. Thus, AlkR protein is produced upon the addition of aTc and the amount of AlkR protein produced is positively correlated with the aTc concentration.

Figure 6 : Y axis corresponds to RFU (Relative Fluorescence Unit) 2 hours after octane addition and on the X axis sample groups are indicated. “(-)” denotes no addition of octane and “(+)” denotes 1 mM octane addition.

Figure 7 : Y axis corresponds to RFU (Relative Fluorescence Unit) 4 hours after octane addition and on the X axis sample groups are indicated. “(-)” denotes no addition of octane and “(+)” denotes 1 mM octane addition.

Figure 8 : Y axis corresponds to RFU (Relative Fluorescence Unit) 16 hours after octane addition and on the X axis sample groups are indicated. “(-)” denotes no addition of octane and “(+)” denotes 1 mM octane addition.

Gradient aTc induction results indicated that the most significant fold change in GFP signal is obtained from the addition of 25 ng/μl and 10 ng/μl aTc.

AlkR/pAlkM Sensor Gradient Octane Induction

To determine the concentration of foctane which would facilitate a higher fold change in GFP signal, gradient octane induction is performed. Since the most appropriate aTc concentration is determined in gradient aTc induction, all samples are initially induced with 10 ng/μl aTc. On the other hand, the least concentrated octane which provides a sufficient fold change, might be detected by gradient octane induction.

Figure 9 : Y axis corresponds to RFU (Relative Fluorescence Unit) 16 hours after octane addition and on the X axis sample groups are indicated. Voc: octane.

According to gradient octane induction results, it can be concluded that 0.01 mM octane has the most significant GFP level raise. However, the GFP signal amplification can be enhanced in further experiments.