Difference between revisions of "Team:NUS Singapore/Notebook/Group3"
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Revision as of 14:07, 19 October 2016
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Wet Lab Notebook: RIOT RESPONDER
Jun
2016
Overall Strategies
1 June, 2016 Milestone 3: : Overall cloning design
July
2016
Construct 2:
Fig 2. A. LuxI gene is cloned from iGEM2016 distribution kit with primers GEM008 and GEM011. The observed band size of LuxI with overhangs is close to the expected band size of 629 bp. B.Terminator BBa_B0015 is amplified using GEM009 and 012 and stitiched with LuxI. C. Promoter-RBS fragment is synthesized and stitiched to LuxI-terminator by PCR reaction. The observed band size for full LuxI construct is close to the expected band size of 936 bp.
4 July, 2016 Milestone 3: : Construction of pLuxR-LuxI-HA
Aug
2016
1 Aug, 2016 Milestone 2: Construct HasA expression plasmid
26 June: RE digested promoter BBa_K525998 plasmid with SpeI and PstI-HF and hasA with XbaI and PstI-HF. Ligated the two fragments overnight at room temperature 27 June: Transform biobrick promoter + hasA ligation into DH5 alpha 28 July: Colony PCR using VF2 and VR primers
29 July: RE digest promoter +hasA with SpeI and PstI-HF and terminator BBa_B0015 with XbaI and PstI-HF. Ligated the two fragments overnight at room temperature. 30 July: Transformed hasA+pro and terminator ligation into DH5 alpha 31 July: Colony screening of complete hasA construct using VF2 and VR Picked colonies 2 and 6 for overnight culture 1 Aug: Miniprepped the overnight culture and sent colony 2 plasmid for sequencing.
28 July: RIOT responder circuit custom synthesised in three plasmids (Seq1, Seq 2, Seq3). PCR of fragments successfully carried out 29 July: Seq3 fragment inserted into Biobrick plasmid pSCB13 via RE digest and ligation 30 July: Seq3 in pSCB13 transformed into E.coli 2 Aug: Colony screening for Seq3 yielded one positive colony 3 Aug: Seq3 pSCB13 extracted and inserted with Seq2 via RE digest and ligation 4 Aug: Seq2+Seq3 in pSCB13 transformed into E.coli 5 Aug: Colony screening for Seq2+Seq3 yielded several positive colonies 9 Aug: Seq2+Seq3 pSCB13 extracted and inserted with Seq1 via RE digest and ligation 10 Aug: Seq1+Seq2+Seq3 in pSCB13 transformed into E.coli 11 Aug: Colonies were picked for overnight culture and Miniprepped 12 Aug: RE digest of extracted Seq1+Seq2+Seq3 pSCB13 yielded several positive colonies
Sep
2016
22 September, 2016 Milestone 4: Induction and Western blot of HasA
14 Sep: Mass liquid culture of HasA bacteria and induction of expression by IPTG 15 Sep: Lysis of HasA culture 17 Sep: Purification of HasA by nickel column 22 Sep: Completed Western blot of HasA
19 Sep: RIOT responder bacteria induced with HasA protein at 10-4 M and 10-5 M. Fluorescence microscopy pictures taken at hourly intervals 20 Sep: Overnight HasA induced RIOT responder bacteria observed under fluorescent microscope
HasA induction of RIOT responder bacteria (overnight) Top: red fluorescence microscopy pictures of (from left to right) negative control, and 10-5 M HasA. Bottom: bright field microscopy pictures of (from left to right) negative control, and 10-5 M HasA.
20 September, 2016 Milestone 6: Induction of RIOT Responder circuit
Oct
2016
13 October, 2016 Milestone 5: Western blot of Has operon construct
6 Oct: 500mL liquid culture of RIOT Responder bacteria 7 Oct: Lysis of liquid culture 11 Oct: Nickel column purification of HasR, HasS, and HasI proteins 12 Oct: SDS PAGE of purification fractions and electrotransfer to PVDF membrane 13 Oct: Western Blot and chemiluminescent visualisation of HasR, HasS, and HasI bands
