Difference between revisions of "Part:BBa K1321335:Experience"

Revision as of 16:57, 24 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1321335

Figure 1 - Congo Red assay, positive colonies

Figure 2 - Congo Red assay, negative control

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.

Figure 3 - Assaying Congo Red (CR) binding by measuring the changes in absorbance at 490nm

User Reviews

UNIQ52e8253fd59ec5cb-partinfo-00000000-QINU UNIQ52e8253fd59ec5cb-partinfo-00000001-QINU

@@ Line 5: / Line 5: @@
 ===Applications of BBa_K1321335===
+[[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_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.
-Part BBa_K1321335 was digested with XbaI and PstI and the resulting fragment, containing the optimised coding sequences of AcsC and AcsD, was 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. For more information on the characterisation procedure, click here: https://parts.igem.org/Part:BBa_K1321334:Experience
+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.
+[[File:LBfraction.png|500px|thumb|right|Figure 3 - Assaying Congo Red (CR) binding by measuring the changes in absorbance at 490nm]]
 ===User Reviews===