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<li> No more backbone PCR amplifications took place this week.</li> </ul> | <li> No more backbone PCR amplifications took place this week.</li> </ul> | ||
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Revision as of 12:44, 31 October 2017
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Glossary
- FNR - Ferredoxin NADP+ reductase.
- Fer- Ferredoxin and Ferredoxin NADP+ reductase (FNR).
- Hyd- hyd 1 coding for the enzymatic part of the Hydrogenase molecular machine.
- Fer/Hyd- Fer and Hyd as described above in a new biobrick construct.
- HydEF- coding two of the three maturation enzymes.
- HydEFG- hyd EF and hyd G, coding all three maturation enzymes in a new biobrick construct.
- Fer/Hyd/EFG, Omega Ω or HGPCC - plasmid with all the necessary genes coding the total Hydrogenase molecular machine. Ferredoxin, Ferredoxin NADP+ reductase, Hyd 1, Hyd EF and Hyd G all in a new biobrick construct. This plasmid gained the official bio-brick name – Hydrogen Gas Producing Gene Cluster (HGPGC).
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![]() 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 ~1600 bp. Lanes 11-14 show bands of ~3000 bp 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 ~1000 bp and ~2000 bp, indicating all restriction enzymes are functional in cutsmart buffer. |
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![]() Figure 2. Agarose gel (1%) electrophoresis of single and double digests using Eco-RI (E) and PstI (P) in fer/hyd DNA 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 (~3400 bp and 2000 bp) 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. |
![]() Figure 3. 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. |
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Wet LabOur plan was as follows:
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![]() Figure 4. 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 ~2700 bp. Lanes 2, 4 and 6 show double digest of fer using Eco-RI and PstI (E+P) with bands at ~1700 bp and ~2000 bp. 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 (~1700 bp and ~2000 bp). |
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![]() Figure 5. 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 (~5300 bp) as well as the bands of the double digests (~3500 bp and ~2000 bp). These bands correspond with the expected weights of fer/hyd1 in CAM. |
![]() Figure 6. 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 ~8700 bp with ~2000 bp 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. |
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![]() Figure 5. 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. |
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![]() Figure 5. SDS-PAGE gel of induced and purified fer fractions. |
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![]() 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. Spectrophotometer at 550 nm 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. |
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