Difference between revisions of "Part:BBa K1927003"
(→Applications of BBa_K1927003) |
|||
| Line 10: | Line 10: | ||
===Applications of BBa_K1927003=== | ===Applications of BBa_K1927003=== | ||
| − | + | The UiO team have submitted a similar part <a href="https://parts.igem.org/Part:BBa_K1927002">BBa_K1927002</a> We therefore looked into the iGEM library and found BBa_J04500 which has a ribosomal binding site and IPTG inducible promotor. By combining AmpR gene downstream for the BBa_J04500 we could theoretically express the gene product which is a β-lactamase class A. This was done by restriction digestion of part BBa_J04500 with Spel and Pstl and the AmpR-insert with Xbal and PstI. Restriction digest was followed by ligation and transformation of TOP10 cells and grown on LB agar plate with chloramphenicol. Colony PCR was performed to verify successful ligation with our insert. Restriction digestion of miniprepped DNA was performed to confirm prior to sequencing. | |
| + | |||
| + | |||
| + | https://static.igem.org/mediawiki/parts/8/88/Pcr003.jpg <br> | ||
Figure3: Gel analysis of colony PCR with BBa_K1927003. The insert was inserted into the shipping vector pSB1C3 by using 2 different approaches; 3A assembly and a single restriction digest followed by ligation. Lane 1 and 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 2 and 11 are positive control (validated part BBa_K1927000). Lane 3-7 are different colonies from 3A assembly. Lane 8-9 and 12-14 are restriction digest and ligation approach. Lane 4, 6-8, 12-13 shows the appropriate band at approx. 1000bp. | Figure3: Gel analysis of colony PCR with BBa_K1927003. The insert was inserted into the shipping vector pSB1C3 by using 2 different approaches; 3A assembly and a single restriction digest followed by ligation. Lane 1 and 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 2 and 11 are positive control (validated part BBa_K1927000). Lane 3-7 are different colonies from 3A assembly. Lane 8-9 and 12-14 are restriction digest and ligation approach. Lane 4, 6-8, 12-13 shows the appropriate band at approx. 1000bp. | ||
Revision as of 11:50, 20 October 2016
Beta - lactamase AmpR
This part is an ampicillin resistant gene with the J04500 inducible promotor.
Usage and Biology
Applications of BBa_K1927003
The UiO team have submitted a similar part <a href="https://parts.igem.org/Part:BBa_K1927002">BBa_K1927002</a> We therefore looked into the iGEM library and found BBa_J04500 which has a ribosomal binding site and IPTG inducible promotor. By combining AmpR gene downstream for the BBa_J04500 we could theoretically express the gene product which is a β-lactamase class A. This was done by restriction digestion of part BBa_J04500 with Spel and Pstl and the AmpR-insert with Xbal and PstI. Restriction digest was followed by ligation and transformation of TOP10 cells and grown on LB agar plate with chloramphenicol. Colony PCR was performed to verify successful ligation with our insert. Restriction digestion of miniprepped DNA was performed to confirm prior to sequencing.
Figure3: Gel analysis of colony PCR with BBa_K1927003. The insert was inserted into the shipping vector pSB1C3 by using 2 different approaches; 3A assembly and a single restriction digest followed by ligation. Lane 1 and 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 2 and 11 are positive control (validated part BBa_K1927000). Lane 3-7 are different colonies from 3A assembly. Lane 8-9 and 12-14 are restriction digest and ligation approach. Lane 4, 6-8, 12-13 shows the appropriate band at approx. 1000bp.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 942