1. Testing for the construct of PET to TPA and EG (Synthesis of Petase)

Experimental Design

Construct Used:

Figure 1: Construct of synthesis of Petase

Purpose:

BBa_K3478888 is the LCC- ICCG gene, which codes for the enzyme Petase that can degrade PET into TPA and EG. To test this construct, the TPA concentration will be tested after the cell is lysed.

Procedure

The procedures for transforming plasmids that either encode the TPA transporter, TPA to PCA construct, PET to TPA, and EG construct are all identical. Our plasmids were all synthesized by Genscript. To transform the cell, we first prepared 250 µL of transformation solution. We then used a loop to transfer colonies of bacteria into the transformation solution. Then, we used another loop to transfer plasmid into the same solution and gently flick it to mix. The solution is then incubated on ice for 10 minutes, and after that transferred to a 42 degrees Celsius water bath for 50 seconds. After that, the tube is put back on ice for 2 minutes. Then, 250 µL of LB Broth was added into the tube, and 100 microliters of the solution was taken out from the tube and plated with a loop. The plate was incubated at 37 degrees Celsius overnight. 30 mL of LB + ampicillin and a colony of bacteria (from the plate using a loop) was then added into the falcon tube. The falcon tube is then incubated in a shaking incubator. The OD 600s are tested every 2 hours and when it reaches a maximum OD600, experiment Group Samples were induced with IPTG (0.1mM and 0.5mM) and a Control group of uninduced cells was also used for comparison. A Blank group containing Untransformed BL21 DE3 cells was also measured. After inducement, the samples are all centrifuged with 5000 rpm for 5 minutes. The supernatant is then taken out for Bradford Assay.

Results

Figure 2: This graph is the standard curve of Bradford Assay with an equation of y= 0.0501x

Calculations of Petase concentration under different time and IPTG concentration:

The Petase synthesized from our engineered E.coli cell has shown to be successfully synthesized and secreted out of the cell. The maximum OD600 level of 0.6 was reached after 240 minutes. From table 2 and figure 10, when the maximum OD600 of 0.6 is reached, the sample with 0.5 mM of IPTG induced has the highest absorbance of 0.19. This means the Petase concentration secreted out of the cell is highest when 0.5 mM of IPTG is induced. Bradford Assay was conducted according to the kit protocol described by Sangon Biotech. Standard curve generated and protein amount in ug/ml was determined as shown in figure 9. Using the standard curve generated from figure 9, the Petase concentration can be calculated in ug/ml. The highest Petase concentration is 3.79 ug/ml when 0.5 mM of IPTG is induced at maximum OD600 (0.6 at 240 minutes). There are also absorbance shown when the IPTG concentration is at 0.0 mM because T7 promoter is a fairly leaky promoter which means even without the use of an inducer, the T7 promoter still expresses the construct to a certain extent (Namdev et al., 2019).

SDS Page:

The measured protein concentration can be further verified as Petase using SDS- Page. The SDS Page is conducted with two samples Control (Not transformed) versus Transformed. The samples are centrifuged, and its supernatant is extracted out. TCA precipitation protocol for concentrating extracellular proteins is used on the supernatants. TCA precipation is required to concentrate the secreted proteins to a detectable level as these proteins are typically very diluted. TCA is used to get 10-50x more concentrated protein.

TCA Protocol:

1. Prepare TCA Solution:
• Add TCA to your culture supernatant to a final concentration of 10-20% (w/v)
• For example: Add 100 μL of 100% TCA to 900 μL supernatant for ~10% final concentration
2. Precipitate Proteins:
• Mix well and incubate on ice for 30 minutes to 2 hours
• Proteins will precipitate out of solution
3. Collect Precipitate:
• Centrifuge at 12,000-15,000 × g for 15 minutes at 4°C
• You should see a protein pellet at the bottom
4. Wash the Pellet:
• Remove supernatant carefully
• Add ice-cold acetone to wash the pellet (removes TCA and salts)
• Centrifuge again at 12,000 × g for 5 minutes at 4°C
• Repeat acetone wash 1-2 more times
5. Dry and Resuspend:
• Air dry the pellet for 5-10 minutes (until acetone evaporates)
• Resuspend in SDS-PAGE loading buffer (much smaller volume than original)

Destaining:

• Transfer the gel into a destaining solution. A common solution is 10% methanol and 10% acetic acid in water.
• Allow the gel to destain for 30 minutes to several hours with gentle rocking. You may need to change the destain solution a couple of times until the background is sufficiently reduced and the protein bands stand out clearly.

Rinsing:

• Once the background has been removed, rinse the gel with distilled water to remove any residual acids and methanol.

2. TPA transporter

Figure 4: Construct synthesizing TPA transporter

Purpose:

This is the construct of TPA transporter in E.coli. BBa_ K4728011 is the tpaK gene that codes for the TPA transporter. The TPA transporter allows TPA to enter the E.coli cell for further processes.

Procedure

The transformation procedures for the cells are the same as the PET to TPA and EG construct, but the plasmid used is the plasmid that codes for TPA transporter. The experimental group is the cells with engineered plasmid of TPA transporter, while the control group is the untransformed cell (wild type BL21 DE3 E.coli cells). The cell is first grown to an OD600 reading of 0.6. Cells are then centrifuged with supernatant removed. Cells were resuspended in M9 minimal media. Experimental Group cells added to M9 media containing TPA (10mM) as the sole carbon source. Control Group Cells added to M9 media containing Glucose plus TPA (10mM) as carbon sources. Afterwards, 0.5 mM of IPTG is used to induce the experimental group only. HPLC is then used to test for the concentration of TPA in the media every 90 seconds.

Results

	Area under the peak
	0 sec	90 sec	180 sec	270 sec
Wild Type	211.582	212.968	205.546	203.046
Transformed Cells	208.247	189.816	154.935	133.299

Table 3: This table is the area under the peak from HPLC for the both samples (wild type BL21 DE3 E.coli cell and transformed cell) at different time

Figure 5: The standard curve for HPLC generated to determine the concentration of TPA in extracellular space with different area under the peak

Figure 6: Line graph of the area under the peak from HPLC for WT vs transformed samples.

Calculations

Wild Type

0 sec:

Y=26.546x-58.272

211.582=26.546x-58.272

X=10.17mM of TPA

90 sec:

Y=26.546x-58.272

212.968=26.546x-58.272

X=10.22mM of TPA

180 sec:

Y=26.546x-58.272

205.546=26.546x-58.272

X=9.94 mM of TPA

270 sec:

Y=26.546x-58.272

203.046=26.546x-58.272

X=9.84 mM of TPA

Experimental Group

0 sec:

Y=26.546x-58.272

208.247=26.546x-58.272

X=10.04 mM of TPA

90 sec:

Y=26.546x-58.272

189.816=26.546x-58.272

X=9.35 mM of TPA

180 sec:

Y=26.546x-58.272

154.935=26.546x-58.272

X=8.03 mM of TPA

270 sec:

Y=26.546x-58.272

133.299=26.546x-58.272

X=7.22 mM of TPA

The TPA transporter from our experiment result has been shown to successfully take in TPA into the cell. As shown in Figure 13 and Table 3, there is a decrease in the area under the peak as the time increases for the transformed sample. The area under the peak of HPLC is a measure of the TPA concentration in the extracellular space of each sample. A decrease in the area under the peak means a decrease in the TPA concentration in the extracellular space. This means the TPA entered the cell via the TPA transporter which resulted in a decrease in TPA concentration in extracellular space. As shown in Figure 13, the area under the peak for the control group (untransformed cell) is approximately constant over time which means the TPA concentration in the extracellular space is constant. This means the TPA did not enter the wild type BL21 DE3 E.coli cell. By using the standard curve shown in figure 12, the actual concentration of TPA in the extracellular space at each time can be calculated as shown in the calculation section. Furthermore, at the fifth time stamp (360 sec), the amount of TPA concentration of the experiment group increased significantly – it was decided to end the experiment and attribute the increase to cell lysis and toxicity of TPA: these cells did not have metabolic pathways to use TPA.

3. PHB to BHB Pathway (Expression of PHAZ_TALFU)

Figure 7: Construct converts PHB into BHB

Purpose

This construct contains PHAZ_TALFU which codes for PHB depolymerase that can break down PHB into BHB which then links with GFP (BBa_K2459011) by a linker (BBa_ K3286268) which allows us to use the measure of fluorescein to find the concentration of BHB synthesized by this construct. This can test the T7 expression system and confirm expression of recombinant proteins.

Procedure

Transformation of BL21 DE3 E.coli cells with the PHB to BHB construct plasmid was done using the same cell transformation procedures that has been used for previous experiments. Four groups were prepared and tested: Wild Type (untransformed BL21 DE3 cells), uninduced transformed BL21 DE3 cells, induced with 0.5mM , induced with 0.1mM. Measurement of GFP expression was used to verify the expression of genes within the system. Under the T7 promoter. IPTG concentrations was used to induce and investigate expression levels. Absolute Fluorescence was measured and graphed. Additionally, MEF (molecules of equivalent fluorescein) was determined for each group using a standard curve (?). We carried out a calibration with fluorescein (from the distribution kit) . We followed the standard protocol as described by the iGEM measurement committee: https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-x54v986dpl3e/v3. Fluorescence was measured using FLUOstar Omega microplate reader.

Results

12 hours
	Fluorescence
	Sample 1	Sample 2	Sample 3	Average
Wild Type (untransformed BL21 DE3 E.coli cells)	186	201	203	197
Uninduced (transformed BL21 DE3 cell)	302	426	412	380
0.5 mM IPTG	702	683	747	711
0.1 mM IPTG	661	641	678	660

13 hours
	Fluorescence
	Sample 1	Sample 2	Sample 3	Average
Wild Type (untransformed BL21 DE3 E.coli cells)	N/A	N/A	N/A	N/A
Uninduced (transformed BL21 DE3 cell)	438	441	424	434
Induced 0.5 mM	742	798	807	782
Induced 0.1 mM	716	709	724	716

14 hours
	Fluorescence
	Sample 1	Sample 2	Sample 3	Average
Wild Type (untransformed BL21 DE3 E.coli cells)	218	221	222	220
Uninduced (transformed BL21 DE3 cell)	477	484	432	464
Induced 0.5 mM	898	709	724	716
Induced 0.1 mM	774	771	836	794

15 hours
	Fluorescence
	Sample 1	Sample 2	Sample 3	Average
Wild Type (untransformed BL21 DE3 E.coli cells)	N/A	N/A	N/A	N/A
Uninduced (transformed BL21 DE3 cell)	514	520	521	518
Induced 0.5 mM	944	932	978	951
Induced 0.1 mM	891	852	911	885

16 hours
	Fluorescence
	Sample 1	Sample 2	Sample 3	Average
Wild Type (untransformed BL21 DE3 E.coli cells)	228	220	219	334
Uninduced (transformed BL21 DE3 cell)	679	673	667	673
Induced 0.5 mM	1015	988	979	994
Induced 0.1 mM	920	917	912	916

Table 4 Fluorescence reading of each group (wild type, uninduced, 0.5 mM IPTG, 0.1mM IPTG) over time with three samples tested for each group at each time period

	Fluorescence
	12 hours	13 hours	14 hours	15 hours	16 hours
Wild Type (untransformed BL21 DE3 E.coli cells)	197	N/A	220	N/A	334
Uninduced (transformed BL21 DE3 cell)	380	434	464	518	673
Induced 0.5 mM	711	782	896	951	994
Induced 0.1 mM	660	716	794	885	916

Table 5 This table shows the average fluorescence at each time and each group for all three samples.

Figure 8: Fluorescein Standard Curve generated using data from Figure 10

Figure 9: Graph showing the average fluorescence over time for each group (wild type, uninduced, induced 0.5 mM IPTG, induced 0.1 mM IPTG)

Figure 10: Table showing the data used to generate the Fluorescein Standard Curve

Calculations

12 hours

Wild Type:

197=3156.2x+14.7

X=0.06 uM

Uninduced:

380=3156.2x+14.7

X=0.12 uM

Induced 0.5 mM IPTG:

711= 3156.2x+14.7

X=0.22 uM

Induced 0.1 mM IPTG:

660=3156.2x+ 14.7

X=0.20 uM

13 hours

Wild Type:

N/A

Uninduced:

434=3156.2x+14.7

X=0.13 uM

Induced 0.5 mM IPTG:

782= 3156.2x+14.7

X=0.24 uM

Induced 0.1 mM IPTG:

716= 3156.2x+14.7

X=0.22 uM

14 hours

Wild Type:

220= 3156.2x+14.7

X=0.07 uM

Uninduced:

464= 3156.2x+14.7

X=0.14 uM

Induced 0.5 mM IPTG:

896= 3156.2x+14.7

X=0.28 uM

Induced 0.1 mM IPTG:

794= 3156.2x+14.7

X=0.25 uM

15 hours

Wild Type:

N/A

Uninduced:

518=3156.2x+14.7

X=0.16 uM

Induced 0.5 mM IPTG:

951= 3156.2x+14.7

X=0.30 uM

Induced 0.1 mM IPTG:

881= 3156.2x+14.7

X=0.27 uM

16 hours

Wild Type:

334= 3156.2x+14.7

X=0.10 uM

Uninduced:

673= 3156.2x+14.7

X=0.21 uM

Induced 0.5 mM IPTG:

994= 3156.2x+14.7

X=0.31 uM

Induced 0.1 mM IPTG:

916= 3156.2x+14.7

X=0.29 uM

	PHB Depolymerase Concentration (uM)
	12 hours	13 hours	14 hours	15 hours	16 hours
Wild Type (untransformed BL21 DE3 E.coli cells)	0.06	N/A	0.07	N/A	0.10
Uninduced (transformed BL21 DE3 cell)	0.12	0.13	0.14	0.16	0.21
UInduced 0.5 mM	0.22	0.24	0.28	0.30	0.31
UInduced 0.1 mM	0.20	0.22	0.25	0.27	0.29

From the experiments, this construct has been proven to successfully express PHAZ_TALFU and synthesize Polyhydroxybutyrate depolymerase. The addition of GFP allows us to measure fluorescence as an indicator for the concentration of Polyhydroxybutyrate depolymerase. As shown in figure 16, the highest concentration of Polyhydroxybutyrate depolymerase is shown to be 0.31 uM when 0.5 mM IPTG is induced for 16 hours and with an induction of 0.5 mM IPTG shows the highest concentration of fluorescence out of all other experimental groups. This means induction wth 0.5 mM IPTG yields the highest concentration of Polyhydroxybutyrate depolymerase.

4. Transformation of Chromoprotein Parts

We first designed parts that included each of the chromoprotein components as described by the 2011 Uppsala iGEM team's collection: http://2011.igem.org/Team:Uppsala-Sweden/2011 
Six different colors were chosen to form the palette for our agar art. These chromoproteins are expressed as proteins inside cells, typically used as reporters in synthetic biology, and are coded for by the following parts:

We placed the coding sequences for the chromoproteins under the control of the pBAD promoter and cloned them into a plasmid containing ampicillin resistance. We selected the pET3a plasmid to achieve strong expression of the chromoproteins, ensuring rich colors for our agar art.
Transformation was carried out using the heat shock method as described at:https://www.addgene.org/protocols/bacterial-transformation/ Cells were grown overnight on selection plates containing ampicillin.

Colonies that grew on the selection plates were inoculated into liquid LB broth containing ampicillin and grown for 12 hours before being transferred onto plates containing LB agar, ampicillin, and arabinose. Results are shown below:

Agar art could now be conducted using our transformed cells engineered to express chromoproteins in the presence of arabinose. Large square plates (9 cm × 9 cm) were prepared with LB agar containing arabinose and ampicillin to ensure chromoprotein expression. Some results are shown below:

References