Difference between revisions of "Part:BBa K4949006"

Revision as of 04:23, 12 October 2023

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 1 and 2 support a functional Lpp-OmpA-RPA1511 surface display mechanism. As observed before, the absorbance at 410 nm increases for treatment reactions with cells containing enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This indicates that our whole cell catalysis approach works. The absorbance at 410 nm for treatment cases containing cells with Lpp-OmpA-RPA1511 increases faster than cases containing cells with Enzymes. These observations suggest two conclusions:

1) Cells with Lpp-OmpA-RPA1511 have enzymes anchoring on the outer membrane and thus can readily access substrates, which enhance catalysis rate and result in a higher rate of increase in absorbance at 410 nm.

2) Cells with Enzymes without anchors can also catalyze cleavage of substrate, unlike our expectation. This suggests that substrates can cross cell membranes to reach enzymes. As cells are pellet before measurement, it also suggests that the product can then cross the membrane again and diffuse into the solution. This hypothesis is further supported by the general higher rate of increase of absorbance at 410 nm when pNOB is used instead of NPO. pNOB has a shorter carbon chain (4) in comparison to NPO (8) and thus, is expected to cross the membrane more readily.

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.

Figure 3 and 4 show that in all cases for Lpp-OmpA-RPA1511, the absorbance at 410 nm increases for treatment reactions with cells decorated with enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This is an indication that enzymes, which are anchored on the outer cell membrane, catalyze the cleavage of substrate into 4-nitrophenol. The rate of increase in absorbance at 410 nm is higher for pNOB substrate than NPO substrate in both enzymes. Furthermore, there is no significant difference between two different cell lines (BL21(DE3) and BL21(DE3) ΔLpp). This indicates the competition between lpp-OmpA-RPA1511 and endogenous genes with Lpp secretion signal for secretion machinery in cells is likely to be not the rate-determining step.

@@ Line 28: / Line 28: @@
 <html><br></html>
-Figure 1 and 2 show that in all cases for Lpp-OmpA-RPA1511, the absorbance at 410 nm increases for treatment reactions with cells decorated with enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This is an indication that enzymes, which are anchored on the outer cell membrane, catalyze the cleavage of substrate into 4-nitrophenol. The rate of increase in absorbance at 410 nm is higher for pNOB substrate than NPO substrate in both enzymes. Furthermore, there is no significant difference between two different cell lines (BL21(DE3) and BL21(DE3) ΔLpp). This indicates the competition between lpp-OmpA-RPA1511 and endogenous genes with Lpp secretion signal for secretion machinery in cells is likely to be not the rate-determining step.
+Figure 1 and 2 support a functional Lpp-OmpA-RPA1511 surface display mechanism. As observed before, the absorbance at 410 nm increases for treatment reactions with cells containing enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This indicates that our whole cell catalysis approach works. The absorbance at 410 nm for treatment cases containing cells with Lpp-OmpA-RPA1511 increases faster than cases containing cells with Enzymes. These observations suggest two conclusions:
+) Cells with Lpp-OmpA-RPA1511 have enzymes anchoring on the outer membrane and thus can readily access substrates, which enhance catalysis rate and result in a higher rate of increase in absorbance at 410 nm.
+) Cells with Enzymes without anchors can also catalyze cleavage of substrate, unlike our expectation. This suggests that substrates can cross cell membranes to reach enzymes. As cells are pellet before measurement, it also suggests that the product can then cross the membrane again and diffuse into the solution. This hypothesis is further supported by the general higher rate of increase of absorbance at 410 nm when pNOB is used instead of NPO. pNOB has a shorter carbon chain (4) in comparison to NPO (8) and thus, is expected to cross the membrane more readily.
 <html><img src="https://static.igem.wiki/teams/4949/wiki/results/figure26-rpa1511-npo-deltalppstrain.png" width="950" height="450"></html>
@@ Line 38: / Line 42: @@
 <html><br></html>
-Figure 3 and 4 support a functional Lpp-OmpA-RPA1511 surface display mechanism. As observed before, the absorbance at 410 nm increases for treatment reactions with cells containing enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This indicates that our whole cell catalysis approach works. The absorbance at 410 nm for treatment cases containing cells with Lpp-OmpA-RPA1511 increases faster than cases containing cells with Enzymes. These observations suggest two conclusions:
+Figure 3 and 4 show that in all cases for Lpp-OmpA-RPA1511, the absorbance at 410 nm increases for treatment reactions with cells decorated with enzymes while the absorbance at 410 nm does not increase notably for control cases for reactions with no whole-cell catalyst. This is an indication that enzymes, which are anchored on the outer cell membrane, catalyze the cleavage of substrate into 4-nitrophenol. The rate of increase in absorbance at 410 nm is higher for pNOB substrate than NPO substrate in both enzymes. Furthermore, there is no significant difference between two different cell lines (BL21(DE3) and BL21(DE3) ΔLpp). This indicates the competition between lpp-OmpA-RPA1511 and endogenous genes with Lpp secretion signal for secretion machinery in cells is likely to be not the rate-determining step.
-) Cells with Lpp-OmpA-RPA1511 have enzymes anchoring on the outer membrane and thus can readily access substrates, which enhance catalysis rate and result in a higher rate of increase in absorbance at 410 nm.
-) Cells with Enzymes without anchors can also catalyze cleavage of substrate, unlike our expectation. This suggests that substrates can cross cell membranes to reach enzymes. As cells are pellet before measurement, it also suggests that the product can then cross the membrane again and diffuse into the solution. This hypothesis is further supported by the general higher rate of increase of absorbance at 410 nm when pNOB is used instead of NPO. pNOB has a shorter carbon chain (4) in comparison to NPO (8) and thus, is expected to cross the membrane more readily.