Team:Macquarie Australia/Results

Aim

We designed and ordered the gBlocks for 4 genes, which encoded for proteins involved in hydrogen production (fer, hyd1, hydEF, hydG) from Chlamydomonas reinhardtii.
Improved gBlock hydG which demonstrated a loss of functionality (2016) due to a point mutation.
These gBlocks were inserted into one Biobrick (known as the Hydrogen Producing Gene Cluster) and transformed into Escherichia coli with a lac promoter and chloramphenicol resistance.
Once induced, we aimed to test the rate of hydrogen gas production in these cells.

Experimental Design

Analyse, optimise and construct the necessary gBlocks.
Digest and ligate gblocks into Biobricks.
Digest/Double digest in conjunction with sequencing to verify gBlocks.
Digest and ligate gBlocks together via standard assembly.
Induce plasmid with IPTG for protein expression.
Run cell lysate of fer on SDS-PAGE followed by Mass Spectrometry to analyse gel bands.
Test hydrogen production using Clark electrode and gas volume measurement experiment.

Summarised Results:

Construction and confirmation of composite parts: hyperlinks to parts registry for fer/hyd1, hydEFG, and Hydrogen Gas Producing Gene Cluster.
Improvement of previous part, hydG, to fix point mutation and provide functionality.
Successful cloning of lac promoters in front of gene constructs.
Confirmed sequencing of parts.
Confirmed transformation into competent cells.
Successful assembly of Omega plasmid in the following order: fer-hyd1-hydEFG. PCR and plasmid double digest confirm the presence of these genes at the expected bands (see Fig. 4).
SDS-PAGE of induced protein expression of ferredoxin and ferredoxin reductase (fer).
Calculated the rate of hydrogen gas production using a Clark electrode which showed 2.5mL of Hydrogen gas was produced per hour.

Results

Verifying Restriction Enzymes and Antibiotic Resistance

The standard restriction enzymes provided by igem (EcoRI, XbaI, SpeI, PstI) were used to digest PCR products to verify they were functional and cut the cellular DNA as expected. An agarose gel electrophoresis (see Fig.1) was performed on digested photosystem II plasmid (psbMZHWK) KODsmart PCR products, as well as chloramphenicol (CAM), ampicillin (AMP) and Kanamycin (KAN) backbones. Enzymes EcoRI (E), PstI (P), XbaI (X) and SpeI (S) were tested. The results show that all digestions cut the psbMZHWK plasmid at the expected ~1000bp mark and appear to be functional in cutsmart buffer. Furthermore, PCR of CAM and AMP was successful, yet Kan was not (see Fig. 1).

Figure 1. Agarose gel (1%) electrophoresis. Lanes 1 and 10 contain a Neb 1kb marker. Lanes 2, and 6 were negative controls. Lane 4 shows no bands and indicates failed PCR of AMP in KOD. Lanes 7-9 show no bands and indicate unsuccessful PCR of respective antibiotics in Quick cells. Lanes 3 and 5 show successful band of CAM and AMP respectively at ~1600bp. Lanes 11-14 show bands of ~3000bp psbMZHWK (PS2) plasmid as expected cut with Eco-RI (E), XbaI (X), SpeI (S) and PstI (P). Lanes 15-18 show successful cut of PS2 plasmid at ~1000bp and ~2000bp, indicating all restriction enzymes are functional in cutsmart buffer.

Following previous verification of PS2 plasmid, the experiment was repeated in an attempt to PCR Kan backbone along with digested CHLP. This agarose gel revealed the same success of functional restriction enzymes cutting CHLP at ~1300bp (see Fig. 2), however Kan PCR remained unsuccessful.

Figure 2. Agarose gel (1%) electrophoresis. Lane 1 contains a 10kb marker. Lanes 2-5 show CHLP with Eco-RI (E), XbaI (X), SpeI (S) and PstI (P) respectively. Lanes 6-9 show restriction enzymes cutting plasmid at~1300bp and all are functional. Lanes 10-11 show unsuccessful PCR of Kan resistance.

Therefore, all restriction enzymes were used throughout our project as the gels showed they were not contaminated and cut test biobricks PS2 and CHLP as expected. Our project focused on using CAM resistance in our biobricks.

fer/FNR characterisation– Electron Transporters Ferredoxin and Ferredoxin Reductase

Following successful screening, the functionality of the fer biobrick was confirmed in our project by purifying out the proteins on a Q column (see Fig. 3). The extracted proteins were then observed with a spectrometre at 550 nm. The results from the spectrometre prove that the fer biobrick has functionality as we observed the reduction of cytochrome C by the oxidisation of NADPH to NADP+ (see Fig. 4). Additionally, an SDS-PAGE gel was run to observe the possible bands corresponding to increased fer proteins when induced with IPTG (see Fig. 5).

Figure 3. Purification of ferredoxin reductase on Q column fractions for samples A, B and C. The intense yellow colouring corresponds with a greater degree of protein purification. Of the 12 fractions taken from each sample, the ones pictured (A10-12, B1, B6-12 and C1) contained purified protein and were used for spectrometry.

Figure 4. Spectrophometre at 550nm of purified ferredoxin reductase (FNR) fraction. The peaks (blue) correspond to the reduction of cytochrome C by the oxidisation of NADPH to NADP+ by FNR. This validates the characterisation of the fer biobrick.

Figure 5. SDS-PAGE gel of induced and purified fer fractions.

fer/hyd1 Assembly - Electron Transporters to Power a Hydrogenase

This biobrick was created to ligate a ferredoxin and ferredoxin reductase (FNR), an electron transporter from NADP+ reduction, to a hydrogenase native in C. reinhardtii. The ferredoxin donates electrons to the hydrogenase for the production of hydrogen gas.
The biobricks fer-FNR (fer –ferredoxin and ferredoxin reductase) and hyd1 ([FeFe] hydrogenase) were screened prior to their assembly by single and double digestions with E and E+P enzymes. Digests were run on agarose gel (1%) and showed appropriate sites were cut in hyd1 (~1700bp) and fer (~1700bp) with a plasmid vector backbone of ~2000bp (see Fig. 6).

Figure 6. Agarose gel (1%) electrophoresis. Lanes 7 and 14 show 1kb marker. Lanes 1, 3, 5, show single digest of fer plasmid using Eco-RI (E) at ~2700bp. Lanes 2, 4 and 6 show double digest of fer using Eco-RI and PstI (E+P) with bands at ~1700bp and ~2000bp. Lanes 8, 10 and 12 show hyd1 single digest with E (~3700bp). Lanes 9, 11 and 13 show double digest of hyd1 using E+P (~1700bp and ~2000bp).

Standard assembly of verified biobricks fer and hyd1 was performed with CAM or AMP resistance. The ligated biobricks were transformed into competent cells and plated onto CAM/AMP plates respectively. Colonies grew, which were further incubated, miniprepped and screened (see Fig. 7).

Figure 7. Agarose gel (1%) electrophoresis of single and double digests using Eco-RI (E) and PstI (P) in fer/hyd1 gene in transformed colony samples A, B, C and D. Lane 1 contains a 1kb ladder. Samples A (lanes 3-4), B (lanes 4-5) and C (lanes 6-7) are from the same transformed plate. Samples A and B show expected band weights for the single digests (~5400bp) and double digests (~3400bp and 2000bp) respectively, and were submitted for sequencing confirmation. Band weights in sample C do not correspond with expected band weights and were unsuccessful. Sample D was spun down prior to loading and no band weights were detected. This gel validates the fer/hyd1 Biobrick to the designed constructs in samples A and B.

Following the validation of the fer/hyd1 biobrick by sequencing, the backbone was swapped to CAM resistance as all other biobricks we created were using this type of antibiotic resistance. The fer/hyd1 backbone was successfully swapped to CAM (see Fig. 8).

Figure 8. Agarose gel (1%) electrophoresis of Biobrick fer/hyd after having the backbone swapped to CAM resistance using digests using Eco-RI (E) and PstI (P). Single (E) and double (E+P) digests were performed. The bands of the single digests correspond to the expected size (~5300bp) as well as the bands of the double digests (~3500bp and ~2000bp). These bands correspond with the expected weights of fer/hyd1 in CAM.

In summary the biobricks fer and hyd1 were successfully ligated together (see Fig. 5), sequencing results confirmed this.

hydEFG Assembly – Hydrogenase Maturation Enzymes

The hydG biobrick was constructed by the 2016 Macquarie iGEM, however it was found to have a 1bp mutation which appeared to cause a loss of functionality. This year we have corrected this mutation and following biobrick creation with transformation into competent DH5α cells, the sequenced results prove we have a functioning maturation enzyme.
This biobrick was ligated with biobrick HydEF to assist in the formation of the H-cluster in the Hydrogenase. Confirmed transformation into competent cells (see Fig. 9) and sequencing means this plasmid will allow the faster assembly of the hydrogenase complex, in turn allowing our cell to produce hydrogen gas sooner.

Figure 9. Agarose gel (1%) electrophoresis of single (E) and double (E+P) digests on colony samples A, B and C. All three samples display expected band weights of ~7500bp for single digests and ~5500bp with ~2000bp double digests. This gel indicates successful ligation of hydG and hydEF biobricks and validates the hydEFG biobrick.

Hydrogen gas producing gene cluster Assembly

With sequencing of the biobricks fer/hyd1 and hydEFG confirmed, all that remained was a final assembly. The Hydrogen Gas Producing Gene Cluster plasmid is a composite part; the total construct of genes fer/FNR/hyd1/hydEFG (see Fig. 10). All promoters are inducible lac promoters with a -35 and -10 consensus sequences of and respectively. The ribosome binding sites had a sequence of following the promoter positioning.

Figure 10. Agarose gel (1%) electrophoresis of transformed Hydrogen Gas Producing Gene Cluster plasmid with single (S-EcoRI-HF) and double (D-EcoRI-HF and PstI) digests. Lanes 2-9 were performed on the 23/8/17 of 4 sample colonies of Quick cells. Lanes 3-5 and 7-9 (samples B, C, D) display expected band weights of ~10,700bp for single digests and ~8700bp with ~2000bp for double digests. Sample A of Quick cells in lanes 2, 6, 13 and 14 did not possess necessary band weights and were discarded. Sample A of commercial cells in lanes 11 and 12 correspond with expected single and double digest band weights. Samples B and C show expected band weights for all single and double digests in both Quick and commercial cells (lanes 15-22). Sample D in commercial cells (lanes 23 and 24) did not possess the expected band weights and were discarded. Sample D in quick cells (lanes 25 and 26) showed the expected band weights for single and double digests. This gel validates the design construct of the HGPGC plasmid.

The fer genes are a ferredoxin and ferredoxin reductase (FNR) involved in the transportation of electrons which are passed to hyd1 (Hydrogenase). The hydEFG genes act as maturation enzymes that aid hydrogenase activation, so that following IPTG induction, when under anaerobic conditions, the gene cluster will begin to produce hydrogen gas.

Achievements:

Constructed recombinant Hydrogen Gas Producing Gene Cluster plasmid with fer/hyd1/hydEFG under lac promoter.
Induced IPTG expression of mature hydrogenase.
Confirmed sequence results for all genes in Hydrogen Gas Producing Gene Cluster plasmid.
Tested the rate of hydrogen gas production using a Clark electrode data.
2.5mL of Hydrogen gas produced per hour in 2mL of induced transformed DH5α cells at 0.1 OD against control group of baseline H2 production.

Summary of Parts

All composite parts underwent single (EcoRI) and double (EcoRI with PstI) digests followed by agarose gel (1%) electrophoresis to summarize and validate the successful construction of the parts comprising the Hydrogen Gene Producing Cluster (see Fig. 11).

Figure 11. Agarose gel (1%) electrophoresis of transformed Hydrogen Gas Producing Gene Cluster plasmid with single (S-EcoRI-HF) and double (D-EcoRI-HF and PstI) digests. Lanes 2-9 were performed on the 23/8/17 of 4 sample colonies of Quick cells. Lanes 3-5 and 7-9 (samples B, C, D) display expected band weights of ~10,700bp for single digests and ~8700bp with ~2000bp for double digests. Sample A of Quick cells in lanes 2, 6, 13 and 14 did not possess necessary band weights and were discarded. Sample A of commercial cells in lanes 11 and 12 correspond with expected single and double digest band weights. Samples B and C show expected band weights for all single and double digests in both Quick and commercial cells (lanes 15-22). Sample D in commercial cells (lanes 23 and 24) did not possess the expected band weights and were discarded. Sample D in quick cells (lanes 25 and 26) showed the expected band weights for single and double digests. This gel validates the design construct of the HGPGC plasmid.

Clark Electrode

Gas Measuring Apparatus

A gas measuring apparatus was utilised to measure the gases produced by 3 different cultures: a culture of untransformed DH5α, one transformed with fer/hyd and one with an induced Hydrogen Gas Producing Gene Cluster. All cylinders were equalised to a starting gas volume of 15 mL. The full methods for the gas measuring apparatus are found here.

Figure //. The overall setup of the gas measuring apparatus. The cylinder is filled with water, up until the 15 mL mark. A glass rod connects the cylinder to the Büchner flask with 80 mL mature culture in M9 media with an OD600 of approx. 0.2, supplemented with 20 mM glucose and 1 mM IPTG. The gas can be removed with a syringe.

The flask was stirred constantly and volume readings for each cylinder were taken at regular intervals. In this apparatus, any gas produced by the cultures displaced water in the cylinder. The modelling results of the apparatus can be found here.

Table 2. Results of rate of gas production (RGP) for the gas measuring apparatus, where the cultures used contained an induced Hydrogen Gas Producing Gene Cluster (HGPGC), an induced Fer/Hyd and DH5α with no insert.
Octopus Replicate 1
Date	Time	Δ GPGC / mL	RGP / mL hr^-1	Δ Fer/Hyd / mL	RGP / mL hr^-1	Δ DH5α / mL	RGP / mL hr^-1
11/10	5pm
12/10	930am	0.95	0.0576	0	0	59.8	3.62
16/10	930am	59.85	3.63	0	0	0.92	0.0558
	5pm	3.8	0.507	0	0	3.68	0.491
Octopus Replicate 2
Date	Time	Δ GPGC / mL	RGP / mL hr^-1	Δ Fer/Hyd / mL	RGP / mL hr^-1	Δ DH5α / mL	RGP / mL hr^-1
18/10	1145am
	6pm	38	6.08	0	0	0	0
19/10	930am	61.75	3.98	3.64	0.235	10.12	0.653
	430pm	17.1	2.14	0.91	0.114	8.28	1.04
	550pm	1.9	1.43	0.91	0.683	0.92	0.690
20/10	915am	20.9	1.36	0.91	0.059	10.12	0.656
	4pm	14.25	4.75	2.73	0.910	3.68	1.23
	545pm	3.8	2.17	2.73	1.56	1.84	1.05
21/10	940am	34.2	2.15	4.55	0.286	22.08	1.39
	440pm	13.3	1.9	0.91	0.13	6.44	0.920
Octopus Replicate 3
Date	Time	Δ GPGC / mL	RGP / mL hr^-1	Δ Fer/Hyd / mL	RGP / mL hr^-1	Δ DH5α / mL	RGP / mL hr^-1
23/10	10am
	12pm	2.85	1.43	2.73	1.37	0	0
	2pm	5.7	2.85	3.64	1.82	0	0
	5pm	3.8	1.27	3.64	1.21	0	0
24/10	8am	28.5	1.9	18.2	1.21	10.12	0.675
	12pm	3.8	0.95	2.73	0.683	3.68	0.92
	4pm	3.8	0.95	2.73	0.683	1.84	0.46
	8pm	1.9	0.475	1.82	0.455	1.84	0.46
25/10	1015am	11.4	0.8	7.28	0.511	5.52	0.387
	12pm	3.8	1.38	0.91	0.331	0.92	0.335
	330pm	1.9	0.543	1.82	0.52	0.92	0.263
	6pm	1.9	0.76	0	0	0.92	0.263
26/10	2pm	17.1	0.855	8.19	0.410	3.68	0.184
	4pm	0.94	0.475	0.91	0.455	0	0

To test for the percent hydrogen gas in each headspace, a 20 mL gas sample was taken from each culture using a syringe and weighed. A sealed tube with 20 mL barium hydroxide was then put under negative pressure and the gas sample was injected and shaken for 10 minutes to remove CO₂ in the form of barium carbonate, a stable precipitate. The remaining gas was extracted back into the same syringe and the volume recorded and again weighed. The same volume of air was then weighed in the syringe, followed by the same volume of pure hydrogen gas (Figure //).

Figure //. Flow diagram of the gas measuring apparatus. The first cylinder starts with a headspace of 80:20 N₂ and O₂. The O₂ is consumed and CO₂ as well as H₂ is produced. N₂ is still present. The CO₂ is extracted and precipitated with BaCO₂. The remaining gases can be weighed to determine the actual concentration of N₂ and H₂. It was determined that 92 percent N₂ and 7 percent H₂ was produced.

After the CO₂ free gas was weighed, when compared with the weight of the free volume of pure H₂ and pure air, the weight of the gas was less than air but more than H₂. This meant that there was a mixture of N₂ and H₂. We estimated there was 50 mL of air present in the start of the apparatus (headspace and tube). Since we can assume that the O₂ was consumed since it is an anaerobic environment, the ratio is of N₂ and O₂ should be 40 mL N₂ and 10 mL O₂. Our actual data showed 92 percent N₂ and 7 percent O₂, which indicated air was leaking in.

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