Part:BBa_K3146010

TodE with promoter and RBS

TodE improved from the previous BioBrick, BBa_K2626000.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 405
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 455
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 480
  

TodE 3-methylcatechol 2,3-dioxygenase enzyme was ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site in order to improve expression. Heat shock transformation protocol was applied, colonies allowed to grow for 24 hours. 4 hour bacterial stock inoculated with single colony was lysed and 2mM of 3-methylcatechol was added to the solution. E. Coli TOP10 and 2mM 3-methylcatechol was used as control. AV at 384 was monitored for 3 hours. Measurements were take in triplicates using Nanodrop 200 Thermo scientific every hour and means were used to construct the plot.

Figure 1

Table1

.

Even though addition of a different RBS and Promoter to the TodE coding sequence could not be directly compared to the previous years results we believe the expression of the enzyme was improved. This statement could be supported due to very similar results being obtained in a different strain of E. Coli top 10 that generally has lower recombinant protein expression than E coli DH-a that was used last year. As well as being able to obtain similar results using 4 hour bacterial stock. Raw data can be seen in table 1 above and plot A.V.agains time can be seen in Figure 1.