Difference between revisions of "Part:BBa K4368001"

Latest revision as of 08:35, 11 October 2022

pcstA + rbs + yebF + cex + terminator

Description

Cex encodes for the exoglucanase gene of Cellulomonas fimi (BBa_K118022). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cenA and bglX. In addition, this part includes the composition used by the team, which includes a strong rbs (BBa_B0030), a double terminator (BBa_B0015) as well as a promoter inducible by glucose concentration (BBa_K118011). Furthermore, we improve this composite adding a gen encoding a motor protein named YebF (BBa_K1610300) that secrete the enzyme out of the E. coli membrane. The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

Congo red assay

A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1044
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 268
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 677
Illegal NgoMIV site found at 1050
Illegal NgoMIV site found at 1552
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1097
Illegal SapI.rc site found at 1180

Contribution

Group: UMA_MALAGA
Author: Molina Calvo, Alonso

@@ Line 4: / Line 4: @@
 ==Description==
-''Cex'' encodes for the exoglucanase gene of ''Cellulomonas fimi'' (<partinfo>BBa_K118022</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cenA'' and ''bglX''. In addition, this part includes the composition used by the team, which includes a strong rbs (<partinfo>BBa_B0030</partinfo>), a double terminator (<partinfo>BBa_B0015</partinfo>) as well as a promoter inducible by glucose concentration (<partinfo>BBa_K118011</partinfo>). Furthermore, we improve this composite adding a gen encoding a motor protein named YebF (<partinfo>BBa_K1610300</partinfo>) that secrete the enzyme out of the ''E. coli'' membrane.
+''Cex'' encodes for the exoglucanase gene of ''Cellulomonas fimi'' (<partinfo>BBa_K118022</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cenA'' and ''bglX''. In addition, this part includes the composition used by the team, which includes a strong rbs (<partinfo>BBa_B0030</partinfo>), a double terminator (<partinfo>BBa_B0015</partinfo>) as well as a promoter inducible by glucose concentration (<partinfo>BBa_K118011</partinfo>). Furthermore, we improve this composite adding a gen encoding a motor protein named ''YebF'' (<partinfo>BBa_K1610300</partinfo>) that secrete the enzyme out of the ''E. coli'' membrane.
 The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.
@@ Line 16: / Line 16: @@
 ===Congo red assay===
-A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 uL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.
+A colorimetric assay for the detection of cellulase activity by bacteria transformed by this plasmid was performed. For this purpose, plates with cellulose-rich medium were created and 50 μL of the transformed bacterial suspension were seeded in the center. After 48 hours, it is developed using a 0.1% solution of Congo red. It is left to incubate in agitation at 400 rpm for 15 minutes. After this incubation time, the plates are washed with 1M NaCl solution and the plates are observed. If cellulose degradation has occurred, an orange halo is created around the bacterial zone, while the rest of the plate will appear reddish. This is because congo red differentially stains cellulose and does not stain cellobiose.
 [[File:congo_red.png|350px|center|]]