Team:NUS Singapore/Notebook/Group2
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Wet Lab Notebook: RIOT RESPONDER
Jun
2016
July
2016
7 June: Immunostaining of MDA-468 cells with Anti-CD44v6
27 June: lysed hela cells (followed cell lysis protocol) 30 June: lysed HepG2 cells (followed cell lysis protocol) 29 June: lysed MDA-468 cells (followed cell lysis protocol) 5 July: lysed MDA-231 cells (followed cell lysis protocol) 5 July: Did SDS-page and Western blot (follow SDS-PAGE and Western blot protocol) 22 July: SDS PAGE and Western Blot of lysed cells 26 July: Completed Western Blot of CD44v6 on Hela, MDA-231, MDA-468 and HepG2
26 July, 2016 Milestone 1: Probing for CD44v6 on Cancer cell lines
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
