Difference between revisions of "Part:BBa K4368003"

Latest revision as of 18:52, 7 October 2022

pcstA + rbs + bglX + terminator

Description

BglX encodes for the β-glucosidase gene of Escherichia coli (BBa_K4368002). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cenA and cex. 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). 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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1609
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1457
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1367

Contribution

Group: UMA_MALAGA
Author: Molina Calvo, Alonso

@@ Line 4: / Line 4: @@
 ==Description ==
-''BglX'' encodes for the β-glucosidase gene of ''Escherichia coli'' (<partinfo>BBa_K4368002</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with th genes cenA and cex. 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>).
+''BglX'' encodes for the β-glucosidase gene of ''Escherichia coli'' (<partinfo>BBa_K4368002</partinfo>). This enzyme is responsible of the degradation of cellulose working coordinated with the genes ''cenA'' and ''cex''. 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>).
 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===
+[[File:bglX.png|350px|center|]]
 <!-- Add more about the biology of this part here
@@ Line 11: / Line 19: @@
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
+==<span class='h3bb'>Sequence and Features</span>==
 <partinfo>BBa_K4368003 SequenceAndFeatures</partinfo>
 ==Contribution ==
-*'''Group:''' [https://2022.igem.wiki/uma-malaga/index.html]
+*'''Group:''' [https://2022.igem.wiki/uma-malaga/index.html UMA_MALAGA]
 *'''Author:''' Molina Calvo, Alonso