Team:IIT Kanpur/Notebook

Design construct for secretion assay : Through July

 

August 28, 2018

 

  1. Transformation of all J. Anderson promoter
  2. RE Digestion of received constructs with XbaI+SpeI for cloning purpose.

 

 

August 30, 2018

 

  1. Mini prep of J23100, J23114, J23102, J23110 (J. Anderson’s promoter)

 

 

 

August 31, 2018

 

  1. RE Digestion (With XbaI+SpeI)  of

pelB  sfGFP

pelB linker sfGFP

ompT sfGFP

ompT linker sfGFP

 

      2.  RE Digestion of Kanamycin backbone with with XbaI+SpeI

      3. Transformation of JA promoter.

 

September 2, 2018

 

  1. RE Digestion of Plasmid Backbone
  2. Ligation of blunt ended Kanamycin backbone of received constructs.

 

September 4, 2018

  1. Transformation of J23105 

 

 

September 7, 2018

  1. Phosphorylation of 5’ ends of

            pelB-linker-sfGFP

            ompT-linker-sfGFP

 

September 8, 2018

  1. Ligation of phosphorylated constructs with Kanamycin backbone.

 

 

September 9, 2018

 

  1. Transformation of ligation performed previous day
  2. Culture of JA23105, JA23102, JA23114, JA23110

 

September 10, 2018

 

  1. Mini prep of JA23105, JA23102, JA23114, JA23110

 

 

September 12, 2018

 

  1. Phosphorylation of pelB-linker-sfGFP and ompT-linker-sfGFP (with changed buffer and new enzymes)

 

September 13, 2018

  1. Ligation of phosphorylated constructs with Kanamycin backbone

 

September 18, 2018

  1. Mini prep of ompT-linker colonies. (Picked from previous ligation)

 

September 20, 2018

  1. RE Digestion of ompT-linker with SpeI to check the enzyme.

 

September 21, 2018

  1. Transformation of SDSa backbone from plasmids gifted by team TecCem
  2. RE digestion repeated (as done on previous day, with new enzymes)

 

September 25, 2018

  1. RE digestion of JA100 pelB linker sfGFP with EcorI and PstI.

 

 

September 26, 2018

  1. Ligation reaction of digested construct in Chloramphenicol resistance containing backbone

 

September 27, 2018

 

1.    Ligation reaction repeated.

 

September 28, 2018

 

1.    Ligation reaction with changed parameters for the same insert and vector as on previous day.

 

September 29, 2018

 

  1. Transformation of both ligations done on previous day
  2. Transformation of plasmid containing SDSa and plasmid containing JA102 promoter
  3. RE digestion of received construct with EcorI and PstI

 

 

 

October 1, 2018

  1. RE Digestion of pSBIK3.m1, pSB1A3 and received constructs from IDT with EcorI and PstI
  2. Ligation reaction of IDT constructs in pSBIK3.m1and pSB1A3

 

 

October 2, 2018

  1. Transformation for the above ligation.
  2. RE Digestion JA102 backbone with EcorI and ligating the linearized template to check whether ligase is working.

 

October 4, 2018

 

  1. RE Digestion of IDT constructs with EcorI and PstI
  2. RE Digestion of template pSBIK3 and pSB1C3 with EcorI and PstI
  3. Transformation of SDSa
  4. Culture of SDSa colonies
  5. Ligation of digested templates.

 

October 6, 2018

  1. Transformation of all constructs in Chloramphenicol and Kanamycin backbones

 

 

October 7, 2018

 

  1. Culture set for SDSa transformed colonies and experiment for quantitating protein secretion from our cloned constructs.
  2. RE Digestion of backbone

 

October 8, 2018

 

  1. Promoter sfGFP analysis of JA102-pelB-ompT
  2. LB chloramphenicol plates were made
  3. Transformation of JA100, JA102, JA105, JA108-pelB and JA102, JA105-ompT containing IDT constructs.
  4. SDSa amplification using PCR
  5. Culture of constructs for shipping
  6. Pancau Staining for checking protein secretion via JA102  pelB/ompT containing backbones

 

October 9, 2018

  1. PCR amplification of SDSa gene.
  2. Mini prep of JA102 pelB, JA105-pelB/ompT containing construct
  3. Interlab experiment doe quantitating protein secretion performed for JA102 pelB/ompT ,
  4. Culture set for other quantitative experiments
  5. Western blot for JA102 pelB/ompT

October 10, 2018

 

  1. All parts were shipped.
  2. Anti GFP staining for JA102/JA105/JA100 pelB, JA102/105 ompT
  3. Fluorescence and OD measurement at different time points for above constructs.
  4. Experiment for effects of SDS on growth of E.coli(DH5 alpha)

 

October 11, 2018

 

  1. Fluorescence and OD measurement at different time points for above constructs.
  2. Western Blot repeated with supernatant only.

October 13, 2018

 

  1. Silver staining of JA102/JA105/JA100 pelB, JA102/105 ompT
  2. Culture set for western blot via anti HIS antibody.

 

 

October 15, 2018

 

  1. Western Blot staining with HIS antibody.
  2. 50ml culture of JA105 pelB containing secretion assay construct.

 

 

October 16, 2018

 

  1. Transformation of all cloned construct in Rosetta strain or E.coli (BL21)
  2. Western blot using anti HIS antibody

 

October 17, 2018

 

  1. Set culture for colonies obtained.

 

 

 

October 18, 2018

 

  1. Ponceau staining of the constructs.
  2. Western blot using anti HIS antibody.