Difference between revisions of "Team:NRP-UEA-Norwich/Results/Cloning"

Cloning FeFe and NiFe Hydrogenases into pSB1C3 from BBa_J04450 Aim: Clone the FeFe and NiFe hydrogenase genes individually into iGEM BioBricks, which can then be cloned into pBAD vector using Golden Gate Cloning (GGC).

Basic cloning techniques were followed see protocols 0.1 to 1.6 for specifics. This included, PCR amplification, restriction digests and ligation into the BioBrick vectors. (See supplementary figure 1 and 2 for PCR and restriction digests). After successful ligations colonies were mini prepped and sent off for sequencing to confirm correct ligation. Individual genes were ligated into pSB1C3, BBa_J04450. Consequently, the individual genes were ligated together into our expression vector: pBAD.

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

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.

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. Sizes for gene inserts (bp) are: 433, 1325 and 764 for HydB C-terminal, HydA and HydC/Fdh respectively. (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. Sizes for gene inserts (bp) are: 1830, 1830 and 754 for HyaB N-terimus, HyaB C-terminus and HyaC respectively. HyaA is on lanes 10 to 12 on figure 1A, size is 1215bp. Ligation reaction products were transformed into E.coli cells. Colonies were picked and boiled for PCR analysis with respective primers at PCR settings according to their melting temperatures. HyperLadder Kb was used for both agarose gels, sizes shown in bp. Agarose gel (1%) was run at 110V for 1.5h.

Based on figure 1, it was confirmed that these are BioBricks. The DNA from the colony in lane 6 (Fig. 1A) i.e. HydA, was submitted to the iGEM registry. The DNA from the colony in lane 7 i.e. HydA, colony of lane 8 i.e. HydC/Fdh and colony of lane 3 i.e. HydB C-terminus from Fig. 1A was used for Golden Gate cloning. HydB N-terminal, was not subjected to PCR colony analysis. However, sequencing data confirmed it correctly ligated into pSB1C3, which was then used in Golden Gate cloning. DNA from the colony of lanes 10, 11, 13, 14, 15 and 17 from Fig. 1B were sent off for sequencing. Due to time constraints ligation of the NiFe hydrogenase construct into the pBAD vector could not be performed.  

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.

Constructs were designed by our advisor to ensure correct order of ligations into the pBAD vector. Consequently, the reaction was set up in the PCR machine according to protocol 1.7. The product was then immediately transformed into E.coli competent cells purchased from Bioline.

Successful transformations were observed as colonies on 26 August 2016. These were then inoculated. Following overnight inoculation, the samples were mini-prepped, see protocol 0.4, and sent for sequencing to Eurofins. An agarose gel was run with the successful constructs against just the pBAD vector, as there should be a difference in size when ligated successfully (Fig. 2)

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.