SNA-GFP and GNA-GFP Construct Design

Both N- and C- terminus fusion strategies were used to assemble constructs of the SNA B- chain (SNA-B) as positive control and GNA sequences as negative control fused to GFP sequence in pGFPuv expression vector. The sequences were both checked for the presence of internal restriction sites and to confirm that the restriction sites used were not present in the sequence using CLC Sequence Viewer program (Qiagen, UK). The cloning map and primers were designed using SnapGene software (SnapGene- USA).

97 3.3.2.1 N-terminus cloning

The N-terminus of SNA-B and GNA sequences were fused to the C-terminus of GFP sequence between SacI:EcoRI and SacI:SpeI restrictions sites respectively as shown in figure 33 and figure 34. The partial sequences of pGFPuv expression vector containing SNA-B and GNA are also shown in figure 35 and figure 36. Primers used to introduce the restriction sites to SNA-B and GNA are shown in Table 14.

Figure 33. Cloned SNA B-chain N-terminally fused to GFP protein sequence in the pGFPuv expression vector. The GREEN and RED parts of the arrow represent GFP and SNA-B sequences respectively; The light green and yellow arrows represent the ampicillin resistant (AmpR) and signal of origin of replication (ori) sequences of pGFPuv vector. .

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Figure 34. Cloned GNA N-terminally fused to GFP protein sequence in the pGFPuv expression vector. The GREEN and RED parts of the arrow represent GFP and GNA sequences respectively; The light green and yellow arrows represent the ampicillin resistant (AmpR) and signal of origin of replication (ori) sequences of pGFPuv vector.

Figure 35 and 36 show a detailed view of the N-terminus cloning strategy, note that the SNA-B and GNA sequences are in frame with the GFP sequence and the stop codon (TAA).

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Figure 35. The full nucleotide and deduced amino acid sequence of SNA-B chain N-terminally fused to GFP between SacI and EcoRI restriction sites in pGFPuv expression vector. The GFP sequence is indicated by a GREEN plot bar; The SNA-B sequence is indicated by a RED plot bar.

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Figure 36. The full nucleotide and deduced amino acid sequence of GNA N-terminally fused to GFP between SacI and SpeI restriction sites in pGFPuv expression vector. The GFP sequence is indicated by a GREEN plot bar; The GNA sequence is indicated by a RED plot bar.

103 3.3.2.2 C-terminus cloning

The C-terminus of SNA-B and GNA sequences were fused to the N-terminus of GFP sequence between HindIII:XbaI and XbaI:KpnI restrictions sites respectively as shown in figure 37 and figure 38. The partial sequences of pGFPuv expression vector containing SNA-B and GNA genes are also shown in figure 39 and figure 40. Primers used to introduce the restriction sites to SNA-B and GNA are shown in Table 14.

Figure 37. Cloned SNA-B C-terminally fused to GFP protein sequence in the pGFPuv expression vector. The RED and GREEN parts of the arrow represent SNA-B and GFP sequences respectively; The light green and yellow arrows represent the ampicillin resistant (AmpR) and signal of origin of replication (ori) sequences of pGFPuv vector.

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Figure 38. Cloned GNA C-terminally fused to GFP protein sequence in the pGFPuv expression vector. The RED and GREEN parts of the arrow represent GNA and GFP sequences respectively; The light green and yellow arrows represent the ampicillin resistant (AmpR) and signal of origin of replication (ori) sequences of pGFPuv vector.

Figure 39, 40below show a detailed view of the C-terminus cloning strategy, note that the SNA-B and GNA sequences are in frame with the start codon (ATG) of the expression vector and with the GFP sequence.

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Figure 39. The full nucleotide and deduced amino acid sequence of SNA-B chain C-terminally fused to GFP between HindIII and XbaI restriction sites in pGFPuv expression vector. The SNA-B sequence is indicated by a RED plot bar; The GFP sequence is indicated by a GREEN plot bar.

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Figure 40. The full nucleotide and deduced amino acid sequence of GNA C-terminally fused to GFP between XbaI and KpnI restriction sites in pGFPuv expression vector. The GNA sequence is indicated by a RED plot bar; The GFP sequence is indicated by a GREEN plot bar.

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Following successful transformation SNA-B and GNA plasmid DNA was extracted and the diagnostic restriction digest of the plasmid DNA was performed to confirm the presence of targeted sequences (Figure 41)

Figure 41. Restriction digest of pGFPuv plasmid to confirm the presence of SNA-B and GNA product inserts. Lane 1: HyperLadder I (Bioline, UK); Lanes 2 and 6: the restriction digests of SNA-B and GNA samples C-Terminally fused to GFP respectively; lanes 4 and 8: the restriction digests of SNA-B and GNA samples N-Terminally fused to GFP respectively; Lanes 3, 5, 7 and 9: the undigested negative controls.

Figure 41 confirmed the predicted band size of N- and C- terminus fusion proteins which are 810bp and 795bp for SNA-B fusions and 487bp and 481bp for GNA fusions respectively.

Several sequencing reactions were also performed to determine the quality of the cloned sequences in the recombinant constructs.Partial results are as shown in figure 42, 43, 44, 45 and the full sequences can be seen in appendix 3.

795bp 810bp 481bp 487bp 400 2000 600 800 1000 3000 1500 200 4000 1 bp 2 3 4 5 6 7 8 9

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Figure 42. Chromatogram showing the sequence quality of SNA B-chain (SNA-B) N-Terminally fused to GFP in pGFPuv vector (Partial sequence data shown). The selected region is representative of overall sequencing quality and full sequence is shown in appendix 3. The Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) codons are in green, black, blue and red colours respectively.

Figure 43. Chromatogram showing the sequence quality of SNA B-chain (SNA-B) C-Terminally fused to GFP in pGFPuv vector (Partial sequence data shown). The selected region is representative of overall sequencing quality and full sequence is shown in appendix 3. The Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) codons are in green, black, blue and red colours respectively.

Figure 44. Chromatogram showing the sequence quality of GNA N-Terminally fused to GFP in pGFPuv vector (Partial sequence data shown). The selected region is representative of overall sequencing quality and full sequence is shown in appendix 3. The Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) codons are in green, black, blue and red colours respectively.

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Figure 45. Chromatogram showing the sequence quality of GNA C-Terminally fused to GFP in pGFPuv vector (Partial sequence data shown). The selected region is representative of overall sequencing quality and full sequence is shown in appendix 3. The Adenine (A), Guanine (G), Cytosine (C) and Thymine (T) codons are in green, black, blue and red colours respectively.

3.3.3 Transformation of BL21(DE3) E. coli Cell Line with pGFPuv Vector