Team:BYU Provo/Notebook/CholeraDetection/Summerexp/Period1/Dailylog

Cholera Detection July-August Notebook: July 1 - July 14 Daily Log

7/1/2013

Plan for Cro- We want to induce lambda phage lysis through over production of Cro. So we put Cro behind the pBAD promoter (inserted Cro into pIG12) and will induce Cro with arabinose. Second step we want to modify the tail fibers of lambda so that a biofilm degrading enzyme will be added.

Construct pJG80 constitutes as follows: HapR/GRP-RFP/Qrr4-Lux0/U Plan for quorum sensing genes 1. Amplify HapA promoter fused to GFP. B1251- forward primer for pHapA, BI252- Reverse fusion for pHapA with GFP (GFP will come from pGLO 2. Digest/ligate/transform pJG80 and construct from step 1 with PstI and EcoRI. 4. Cut out CqsS from pIG78 and insert into newly formed plasmid.

PCR for pHapA (BI251/BI252) amplified from cholera worked. Amplification of CqsS (BI230/BI231) also worked.

KP,KK,CH

7/9/13
Plan for growing phage: Make overnights of our lambda containing strains. Combine 0.5ml of the overnight with 4ml of top agar (+&- arabinose). Plate onto LB-Amp (+&- arabinose). Top agar- Make 500ml of LB add 8g of agar then autoclave.

More planning for modifying our set of quorum sensing genes to make the simplest circut. 1. Phusion PCR of RFP with Qrr4 promoter 2. Digest pIG78(about 5,300bps) with HindIII/XbaI/BamHI to get LuxO/U/CqsS. Also digset pLAT (pIG12) with HindIII/XbaI. Ligate LuxO/U/CqsS into cut pIG10 and transform.

KK, KP, CH

7/10/13
Set up PCRs- promoter Qrr4 forward (BI223) with RFP fusion (B224). The whole Qrr4 gene with its promoter (BI226/BI227). Both reactions had cholera as template. Both worked. Cleaned them up.

KK, KP, CH

7/12/13
Set up soeing PCR- as primers used pQrr4/RFP from 9/10 and BI225 (reverse RFP). Template: mCherry. Didn't work. Maybe a problem with the template.

KK, KP, CH