Difference between revisions of "Part:BBa K1321335:Experience"
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BBa_K1321335 was digested with XbaI and PstI and cloned into a medium-to-low copy number plasmid (pSB3K3) containing the inducible pLAC promoter. The resulting construct, verified by GreenTaq Colony PCR, was subsequently sub-cloned into BBa_K1321336-containing electrocompetent cells. These were plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification. | BBa_K1321335 was digested with XbaI and PstI and cloned into a medium-to-low copy number plasmid (pSB3K3) containing the inducible pLAC promoter. The resulting construct, verified by GreenTaq Colony PCR, was subsequently sub-cloned into BBa_K1321336-containing electrocompetent cells. These were plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification. | ||
| − | + | [[File:YEAH3.jpg|200px|thumb|left|Figure 1 - Congo Red assay, positive colonies]][[File:YEAH2.jpg|200px|thumb|left|Figure 2 - Congo Red assay, negative control]] | |
BBa_K1321336 was characterised in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (optimised coding sequences submitted as Part BBa_K1321335) cloned into a medium-to-low copy number plasmid, pSB3K3. The functions of AcsC and AcsD are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. | BBa_K1321336 was characterised in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (optimised coding sequences submitted as Part BBa_K1321335) cloned into a medium-to-low copy number plasmid, pSB3K3. The functions of AcsC and AcsD are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. | ||
Cellulose production was assayed by plating transformed cells on Congo Red assay plates containing 20uM CR, 0.5mM IPTG, 0.1% Arabinose, 1% Glucose, 25ug/ml Chloramphenicol and 25ug/ml Kanamycin. Cellulose-producing E.coli colonies turned red in the presence of CR. | Cellulose production was assayed by plating transformed cells on Congo Red assay plates containing 20uM CR, 0.5mM IPTG, 0.1% Arabinose, 1% Glucose, 25ug/ml Chloramphenicol and 25ug/ml Kanamycin. Cellulose-producing E.coli colonies turned red in the presence of CR. | ||
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===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 16:59, 24 October 2014
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Applications of BBa_K1321335
BBa_K1321335 was digested with XbaI and PstI and cloned into a medium-to-low copy number plasmid (pSB3K3) containing the inducible pLAC promoter. The resulting construct, verified by GreenTaq Colony PCR, was subsequently sub-cloned into BBa_K1321336-containing electrocompetent cells. These were plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification.
BBa_K1321336 was characterised in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (optimised coding sequences submitted as Part BBa_K1321335) cloned into a medium-to-low copy number plasmid, pSB3K3. The functions of AcsC and AcsD are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. Cellulose production was assayed by plating transformed cells on Congo Red assay plates containing 20uM CR, 0.5mM IPTG, 0.1% Arabinose, 1% Glucose, 25ug/ml Chloramphenicol and 25ug/ml Kanamycin. Cellulose-producing E.coli colonies turned red in the presence of CR.
User Reviews
UNIQ27a5552842dd11b2-partinfo-00000000-QINU UNIQ27a5552842dd11b2-partinfo-00000001-QINU