Part:BBa_K2122000

Device expressing the Gb3 Synthase enzyme under control of an arabinose inducible promoter

Experimental Validation

Throughout our project, we have successfully transformed E. coli competent cells and validated the expression of the Gb3 synthase mRNA from the arabinose inducible part using RT-PCR and PCR amplification. The RNA was extracted from the induced colonies and the non-induced colonies as a negative control. RT-PCR was performed and the RNA was converted into DNA. DNA amplification was done by PCR with three sets of forward and reverse primers. Amplified products were un on a 1% agarose gel with sybere green staining and visualised under blue light excitation Figure 1 shows colony growth of appropriate selective media. Bands corresponding to amplified Gb3 synthase products resulting from the three different sets of primers used are shown in Figure 2 .

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The primers used are as follows:
Set 1
Forward: 5- GGA GAA AGG GCA GCT CTA TAA C -3
Reverse: 5- CGG ATG GAA CAC CAC TTC TT -3

Set 2
Forward: 5- CCT GTT CAT CAT CGG CTT CA -3
Reverse: 5- AAG TCC GTG TCC AGG TAG AT -3

Set 3
Forward: 5- ATC TAC CTG GAC ACG GAC TT -3
Reverse: 5- TGG CTC TTC TTG TTC CAC AC -3

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1910
  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 2193