Overview

For our lab experiences, we’ve encountered an enormous number of failures which brought us up to the point we are now at; having an extreme feeling of joy, content and tiredness.

Summary of Achievements in our experiments

• We have successfully cloned 5 of our parts to pSB1C3 backbone in order to submit to the registry; and 3 of them to pSB1A3 backbone for our biochemical assays.
• We have confirmed that our insertions and transformations were correct through PCR check.
• We’ve conducted a biochemical assay in which we verified our hypothesis and proved the improving effects of our genes.

Clonings:

In our project, our aim was to increase the tolerance and resistance of KO11, ethanologenic strain of E.coli, to byproducts and inhibitors that occur during bioethanol production, specifically during the pretreatment of lignocellulosic biomass. In order to achieve that, we picked our genes of interest as FucO and GSH.

We’ve designed 5 parts, 2 of which are basic and 3 are composite.

One of our basic parts included only FucO and the other only GSH as the protein coding region; and our composite parts were designed to include GSH and FucO separately and simultaneously.

As a part of our lab journey, we’ve done 8 clonings in total. 5 of them were to be submitted in the registry and 3 of them were for our biochemical assays.

In order to be able to submit our parts to the registry, we inserted all of our parts to pSB1C3 backbone and cloned them to DH5α.

DH5α Clonings:

Clonings of basic parts: FucO only and GSH only.

In order to be able to grow colonies of DH5α with our first and second basic parts, we’ve ligated them to pSB1C3 backbone with a ratio of 1:3. Then, we’ve transformed the ligations to DH5α competent cells and grew colonies on LB agar plates containing chloramphenicol at a final concentration of 40 µg/ml. After obtaining DH5α colonies, we’ve done colony PCR and considered the results: We’ve come to the conclusion that colony no. 4 from FucO and no. 8 from GSH have given the best results.

Thus, we’ve done plasmid isolation for basic 1 from the 4th colony and for basic 2 from the 8th colony, followed by a PCR to confirm our results.

Basic part 1 (FucO) in pSB1C3 (BBa_K2571000) PCR Confirmation:

In the gel image below, the band on the 5th well demonstrates our correctly inserted gene in pSB1C3 backbone. In PCR, we’ve used FucO left and VR primers to check the orientation of our part in backbone. Expected band length to see was 625 bp and the results were as expected.

Basic part 2 (GSH) in pSB1C3 (BBa_K2571001) PCR Confirmation:

10th well in the gel image below, which belongs to the 8th colony from basic 2 (GSH) cloned DH5α, proves our insertion and transformation right. We’ve used GSH specific primers in PCR and expected to see a band of 225 bp.

In order to be able to grow colonies of DH5α with our first, second and third composite parts; we’ve initially ligated the parts to pSB1C3 backbone. composite part 1’s insert to vector ratio was 1:3, composite 2’s was 1:2 and composite 3’s (Bio-E) was 1:1,5. After that, we’ve transformed the ligations to DH5α competent cells and grew the colonies on LB agar plates containing chloramphenicol at a final concentration of 40 µg/ml. After obtaining DH5α colonies, we’ve done colony PCR and considering the results that we obtained, we’ve decided the best outcomes were from the 3rd colony from composite 1, 6th colony from composite 2 and 3rd from composite 3.

Composite part 1 (FucO) in pSB1C3 (BBa_K2571003) PCR Confirmation:

The gel image below belongs to the PCR confirmation of FucO composite part insertion. After plasmid isolation, the PCR we’ve conducted with FucO left and VR primers, using the plasmid as template, was to check orientation of our part in backbone. The expected band length was 754 bp, we decided that 6th and 7th wells confirm our transformation.

Composite part 2 (GSH) in pSB1C3 (BBa_K2571005) PCR Confirmation:

We’ve conducted a colony PCR with GSH specific primers to test our transformations of composite part 2 in DH5α. We wanted to see a band of 225 bp on gel and all GSH colonies have given the band as expected. Below, wells from 11 to 17 confirm our transformations were successful and the 6th colony has given the best result.

Composite part 3: Dual Insertion of FucO and GSH in pSB1C3 (BBa_K2571006) PCR Confirmation:

Our composite part 3, which makes up Bio-E, has the genes both FucO and GSH as coding regions and was successfully inserted in the pSB1C3 backbone. After transforming the plasmid to DH5α competent cells, we’ve conducted the PCR with FucO specific primers and our expected band length for confirmation was 194 bp. All the results seen on the gel (wells 3-11) were positive, proving that our clonings were successful and we have come to the conclusion that the best band was shown by the 3rd colony.

KO11 Clonings:

In our biochemical assays, our aim was to see the effects of our parts on ethanol production, cell growth, and life span using E. coli ethanologenic strain KO11. Since KO11 itself had chloramphenicol resistance in its genome, we had to create a distinction by adding another antibiotic resistance. Thus, we inserted our composite parts to pSB1A3 backbone (carrying Ampicillin resistance) as well and did the transformations to KO11.

First composite part’s insert to vector ratio was 1:3, second part’s was 1:2, and the third part’s was 1:1,5. After that, we’ve transformed the ligations to KO11 competent cells and grew the colonies on LB agar plates containing chloramphenicol at a final concentration of 150 µg/ml and ampicillin at a final concentration of 100 µg/ml. After obtaining KO11 colonies, we’ve done colony PCR and in the end, we recultured the best result giving colonies for plasmid isolation. Chosen colonies were the 3rd colony for both composite part 1 and 2; and the 2nd colony for composite part 3.

Figure 11: Plate image showing composite part 1 (FucO) and composite part 2 (GSH) transformed into KO11. The medium was LB with Chl 150 and Amp 100. Figure 12: Plate image showing composite part 3 (FucO and GSH together) transformed into KO11.The medium was LB with Chl 150 and Amp 100.

Composite part 1 (FucO) in pSB1A3 PCR Confirmation:

After plasmid isolation, PCR with FucO left and VR primers was conducted to check the orientation of our composite part 1 to pSB1A3 backbone, and the expected band length for that confirmation was 754 bp. 8th well in the image below (obtained from the plasmid isolation of the 3rd colony) confirms the orientation.

Figure 13: Gel image of the PCR confirmation of composite part 1 (FucO) insertion into pSB1A3.

Composite part 2 (GSH) in pSB1A3 PCR Confirmation:

We’ve conducted colony PCR with GSH specific primers for our composite part 2 in pSB1A3 backbone. We wanted to see a band of 225 bp on gel and GSH colonies 3, 4 & 5 have given the bands as expected. Below; wells 5, 6 and 7 confirm our transformation and we choose to proceed to the plasmid isolation with the colony number 3.

Figure 14: Gel image of the PCR confirmation of composite part 2 (GSH) insertion into pSB1A3.

Composite part 3 (FucO&GSH) in pSB1A3 PCR Confirmation:

After successfully inserting our composite part 3 (FucO and GSH together) into the pSB1A3 backbone, we’ve done colony PCR with FucO specific primers. Our expected band length for confirmation was 194 bp and all the results came out positive (seen in wells 12-20), confirming our transformation. Since the best result was seen on colony number 2, we’ve done the plasmid isolation from that colony.

Figure 15: Gel image of the PCR confirmation of composite part 3 (dual expression of FucO and GSH) into pSB1A3.

Biochemical Assay

We designed our biochemical assay in order to evaluate the effects of our circuits on life span, cell mass, and ultimately the bioethanol yield of ethanologenic E. coli strain KO11. We carried out two experimental test sets (1. set: 10mM furfural addition; 2. set: 20mM furfural addition) simultaneously.

In our biochemical assay, we had four KO11 ethanologenic strains of E.coli cultured groups to test.

#1 KO11 un-engineered
#2 KO11 with only FucO
#3 KO11 with only GSH
#4 KO11 with Bio-E

Assay #1

Figure 16: Representation of our biochemical assay #1

Throughout our first assay, each group was grown in LB mediums containing 2% glucose and antibiotics.

To culture group #1 (KO11 un-engineered), we only added Chloramphenicol at a final concentration of 40µg/mL since it only had resistance to Chloramphenicol in its genome; and to the mediums of the groups numbered 2, 3 and 4, we added Chloramphenicol at a final concentration of 40µg/ml and 100µg/ml Ampicillin. The reason was; groups 2, 3 and 4 had plasmids which carried Ampicillin resistance due to their backbone (pSB1A3). Thus, with the addition of antibiotics to the mediums, selectivity was assured.

Cultures were grown overnight, and refreshed in the morning. After approximately two hours of incubation, furfural was added to the mediums.

We added furfural at a final concentration of 10 mM to the four test groups’ mediums in our first set and took OD measurements at Abs 600 nm with 1/10 dilution in 24 hour time intervals.

10 mM furfural OD measurements: Abs 600 nm (1/10 dilution)

	24hrs	48hrs	72hrs
KO11 un-engineered	0.52	0.46	0.40
KO11 with only FucO	0.5	0.5	0.43
KO11 with only GSH	0.6	0.53	0.44
KO11 with Bio-E	0.26	0.45	0.46

Figure 17: Graph showing cell growth of assay groups with respect to time.

Analysis of data:

Our data demonstrated that group #1 had a decrease in cell mass throughout the time verifying the inhibition of cell growth in the presence of furfural in the field. Group #2 (KO11 with only FucO) obviously gave better results with respect to un-engineered KO11. However, although the KO11 group with only FucO showed durability and stability in the first 48 hours, it also experienced depletion in cell mass after the 48th hour. This proves that only the presence of the gene FucO in the bacteria weren’t enough avoid cell mass depletion in the long term and is in need of another gene for increased tolerance. Group #4 (KO11 with both FucO and GSH) gave measurement results as we hypothesized by continuing cellular growth in the first 48 hours and maintaining it even after the 48th hour, though at a lower rate. Overall, we can infer that our best part design (Bio-E) was successful enough to battle with the inhibitive effects of furfural.

To gather more information to prove our hypothesis, we designed our second experimental set and added furfural at a final concentration of 20mM to the four test groups’ mediums followed by OD measurements at Abs 600 nm with 1/10 dilution in 24 hour time intervals.

20 mM furfural OD measurements: Abs 600 (1/10 dilution)

	24hrs	48hrs	72hrs
Un-engineered KO11	0.67	0.54	0.60
KO11 with FucO	0.54	0.43	0.61
KO11 with GSH	0.61	0.60	0.58
KO11 with Bio-E	0.29	0.55