Team:NRP-UEA-Norwich/Results/Cloning
Cloning Results
Cloning the FeFe and NiFe hydrogenase genes individually into pSB1C3, which can then be cloned into pBAD vector using Golden Gate Cloning (GGC).
Table 1: Gene fragments used as inserts into pSB1C3 using BBa_J04450 as the source for the vector. Gene fragments were ordered from idtDNA. The sequences of the gene fragments were obtained from GenBank. pSB1C3 BBa_J04450 was provided by the iGEM distribution kit.
| Gene fragments | Size (bp) |
|---|---|
| HyaA | 1215 |
| HyaB-Nterm | 1830 |
| HyaB-Cterm | 1830 |
| HyaC | 754 |
| HydA | 1325 |
| HydB-Nterm | 433 |
| HydB-Cterm | 433 |
| Fdh/HydC | 764 |
Figure 1: PCR Colony analysis of FeFe and NiFe Hydrogenase subunits. (A) PCR Colony of FeFe Hydrogenase subunits. Lanes 2 to 5 have HydB C-terminally tagged gene insert. Lanes 6 and 7 have HydA gene insert. Lanes 8 and 9 have HydC/Fdh gene insert. (B) PCR Colony of NiFe Hydrogenase subunits. Lanes 9 to 11 have HyaB N-terminally tagged gene insert. Lanes 12 to 14 have HyaB C-terminally tagged gene insert. Lanes 15 to 17 have HyaC. HyaA is on lanes 10 to 12 on figure 1A, size is 1215bp. Ligation reaction products were transformed into E.coli cells. HyperLadder Kb was used for both agarose gels, sizes shown in bp. Agarose gel (1%) was run at 110V for 1.5h. See table 1 for gene sizes
By analysing figure 1, it was confirmed that the genes were incorporated into the BioBrick backbone. HydB N-terminal was not subjected to colony PCR analysis. However, sequencing data confirmed correct ligation into pSB1C3, which was then used in Golden Gate cloning.
Cloning FeFe, HydABC, into the pBAD expression vector
Aim
Ligate the individual FeFe genes into the pBAD expression vector using Golden Gate Cloning, which can then be used for downstream expression trials.
The primers for golden gate sequencing were designed to ensure the correct order of parts when ligating into the pBAD vector. The reaction was set up using the corresponding protocol and the thermocycler used to cycle at the correct temperatures. The product was then immediately transformed into α-select E.coli competent cells purchased from Bioline. Golden Gate cloning utilises type IIS instead of type IIP restriction enzymes. Type IIS restriction enzymes are those that recognise a DNA sequence outside of the cut site. This can be customised to be non-specific, allowing for custom overhangs. This means that during ligation between the inserts and their vector, the recognition site for these enzymes will be removed, eliminating the scarring that occurs during cloning with conventional restriction enzymes. These overhangs can be made to ligate consecutive inserts together. A primer was also designed that would guide the correct ligation of the inserts. The primer would be complementary to the consecutive gene inserts, restriction site and their BsaI cut-sites (Kirchmaier and Wittbrodt, 2013). For our experiment we needed the inserts to ligate as: Fdh/HydC, HydA, and HydB C-terminus or Fdh/HydC, HydA and HydB N-terminus. When ordering the gene inserts, the BioBrick pre- and suffix were included, as well as the Golden Gate sequences that would allow for scarless ligation.
Figure 2: Agarose gel of Golden Gate Cloning Transformations of the FeFe hydrogenases into the pBAD vector. 8A5 cluster consists out of Fdh (606), HydA (607) and HydB C-terminus (608). 9A5 cluster consists out of Fdh (606), HydA (607) and HydB N-terminus (609). Total size of both 8A5 and 9A5 is 7022bp, this includes the pBAD vector which is 4500bp. Gel electrophoresis with 1% agarose run at 110V for an hour. Ladder used was HyperLadder Kb.
Successful transformations were observed. These were then used to inoculate 10ml LB with kanamycin. Following overnight incubation, the samples were mini-prepped as described in the protocol, and sent for Sanger sequencing . An agarose gel was run with the successful constructs and the pBAD vector as a control, as there should be a difference in size if the ligations were successful (see Fig. 2)
Kirchmaier S, Lust K, Wittbrodt J (2013) ‘Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs’. PLoS ONE 8(10): e76117. doi:10.1371/journal.pone.0076117
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