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).
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.
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).
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).
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).
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).
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.
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
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:
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).
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.
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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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