Team:Nevada/Week 18

September 17

Joe:

purify protein from 10, 10ml tubes

incubate with rice for 5 hours

September 18

Joe: RFP-SBP ligation for submission

   culture more RFP-SBP in Top10 cells
   Successful sticky of protein to rice, got pictures

Michelle:

   Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM. 

Justin and Dafne Express more SBP-B12 protein

   Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells
   incubate overnight

September 19

Joe: Transform RFP-SBP for submission

Justin and Dafne

   Rinse plate 5x using PBS-T with 5% non-fat milk
   Add 100 ul of blocking buffer (PBS-T with 5% non-fat milk) to 10 wells with protein as well as 10 wells with no protein (control)
   Incubate overnight
   Purify yesterdays protein expression using amylose column (as done before)
   Digest SBP, B12, SBP-B12, SBP-B12 in expression plasmid by EcoRI HF and Pst I HF

September 20

Joe: Culture more RFP

   Digest SBP by EcoRI/PstI for submission
   RFP-SBP: make 10 tubes from glycerol stock
   Colony PCR of RFP-SBP for submission

Michelle:

   PCR colony check M2-BL21 with Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing brightest colony with 100ml LB AMP-CM and making glycerol stocks.

Results: PCR colony was very successful showed that both colonies in lanes #2 and #3 had bright bands [Gel #198]. Therefore indicating large scale culturing could be proceeded.

Justin and Dafne

   Add 10 ul of vitamin B12-HRP to 1 ml of PBS-T
   Make 4 more 100 fold dilutions of above solution
   Add each diluted solution to 2 wells
   incubate overnight
   Run Western blot analysis and Coomassie stain analysis of purified protein from yesterday
   Ligate yesterday’s digestions into submission plasmid, transform into TOP10 cells

Jeremiah & Chris: · Express the TBP+ gene using an L. Arabinose gradient · Digest TBP, SBP, TBP+, TBP+++, and LA-TBP+ o Run on gel (gel #200) · Ligate the genes above into pSB1C3 · Make glycerol stock of LA-TBP+ in BL21 cells

September 21

Michelle:

   Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI, followed by ligation into PSBC13 (digested with EcoRI and PSTI),  and transformation onto LB-CM plates. 

Results: LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM into PSBC13 for iGEM submission.

Justin and Dafne

   Rinse wells with PBS-T 5x
   Rinse well with PBS 3x
   Add TMB-Microwell peroxidase to each well
   Digest plasmids again

Jeremiah & Chris: · Purify pSB1C3 again via glass milk purification o Run on gel (gel #203) · Dephosphorylate pSB1C3