Team:UCLA/Notebook/Recombinant Expression

iGEM UCLA




Recombinant Silk Functionalization

=Raw lab notebook entries= {{#calendar: year=2015 | month= may | title = Team:UCLA/Notebook/Recombinant_Expression | query=preload=Template:UCLA}} {{#calendar: year=2015 | month= jun | title = Team:UCLA/Notebook/Recombinant_Expression | query=preload=Template:UCLA}} {{#calendar: year=2015 | month= jul | title = Team:UCLA/Notebook/Recombinant_Expression | query=preload=Template:UCLA}} {{#calendar: year=2015 | month= aug | title = Team:UCLA/Notebook/Recombinant_Expression | query=preload=Template:UCLA}} {{#calendar: year=2015 | month= sep | title = Team:UCLA/Notebook/Recombinant_Expression | query=preload=Template:UCLA}} Week 7 05/11 – 05/15 Monday 05/11 Received sequencing primers from IDT Plated glycerol stocks of successful transforments for large scale expression Tuesday 05/12: Plating successful with about 10 colonies. Showed successful transformation. I then picked one colony and inoculated 10 ML LB. Added Chlor at 1000X dilution. Grew overnight at 37 Wednesday 05/13: Starter culture grew. Inoculation method is as follows: grew 2 150 mL cultures at 30 and 37C respectively. Added 5mL of starter culture to each. Added Chlor at 1000X dilution. Added IPTG right away at 0.5mM concentration. This is non ideal but worked with my time constraints. This also correlates with expression protocol I used in Yeates' lab. Inducing right away decreases the time we need to be in lab. I will compare the effectiveness of this method to other methods in the coming weeks. I will also compare the diff growth temperatures with both methods. Thursday 05/14: Both of the cultures grew. Spun them down for 15 min at 5000g. Got a hard pellet of good size. Froze at -80 until next week. Must discuss lysis and purification methods.
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Today we began cloning our GFP.

  • PCR'd off template
  • Ran gel
  • Restriction digest
  • Ligated into backbone

For our ligation, we made the following modifications:

  1. Tried it with newly bought ligase
  2. Left reaction overnight instead of 2 hrs
  3. Vector to insert ratio was 1:5 instead of 1:3

PCR Reaction:

Component Volume
5X Q5 Reaction Buffer 5
10 mM dNTPs 0.5
10 uM Forward (primer 3/7) 1.25
10 uM Reverse (primer 8) 1.25
Template (diluted to 1ng/uL) 0.5
Q5 High Fidelity DNA Polymerase 0.25
Nuclease Free Water 16.25

Gel: Lot of bands, all at correct sizes

Goals

Julian's Goals

  • Finish Purification of ABD containing construct
  • Create hydrogel with ABD and B.mori silk and see if it retains/binds albumin

Achievements

Julian's Achievements

  • Cloned the ABD construct and begun purification methods

What to accomplish next

Julian

  • Test albumin binding properties of the ABD silk construct
  • test hydrogel formation and albumin retention

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