Part:BBa_K4949006

RPA1511

RPA1511 is a carboxyl esterase originally identified in Rhodopseudomonas palustris. RPA1511 has been shown to degrade a variety of polymers, including PLA, with an optimal temperature range of 50-60℃. Our team modified the RPA1511 genetic sequence to include the lpp-OmpA anchor in order to characterize the surface-display mechanism.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 279
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 105
  Illegal NgoMIV site found at 114
  Illegal NgoMIV site found at 340
  Illegal NgoMIV site found at 514
  Illegal NgoMIV site found at 574
  Illegal AgeI site found at 139
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 817
  

Figure 1. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with a Lpp deficient plasmid, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 2. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3) + RPA1511(ΔLpp), bacteria with a Lpp deficient plasmid, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 3. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp +RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant was measured at 410 nm.

Figure 4. Absorbance as a function of time for the whole cell esterase activity assay of RPA1511. After induced expression, E.coli BL21 (DE3) + RPA1511 or E.coli BL21 (DE3)ΔLpp + RPA1511, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD₆₀₀=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.