Team:Caltech/Week 12

Monday 8/30

• Use a different plate of L62-1 cells (HSP-LacI-GFP), pick 3 different colonies and re-perform the HSP-LacI-GFP
  • Only use 0 mM Glucose and 5 mM Glucose
• Make new digests of L54 (HSP-LacI-RBS), E0430 (EYFP), and RFP-TetR backbone
• Ligate L54 to L56 (Lysis-Terminator), L54 to L57 (Another Lysis-Terminator), and L54 to E0430
• Transform aforementioned ligations into cells, along with L90 and L91 (both HSP-Lysis)
• Attempt PCR extraction and amplification of E. coli sigma 32 (HtpR) using Colony PCR protocol

Tuesday 8/31

• Fluorescence spectrometry shows that cells in 5 mM glucose produces significantly less GFP than those in no glucose growth medium.
• Transformations of L90-L95 grow slowly in 30 Celsius conditions; no colonies yet
• Ran gel electrophoresis of E. coli sigma 32 PCR product
  • PCR product the right size! Order primers to turn sigma 32 (HtpR) into an RFC 10 BioBrick
• Completed first part in ligating L36 and L37 by PCR

Wednesday 9/1

• Colonies appeared on L91 and L95 plates
• Colony PCR of L91 and L95
• Ran gel electrophoresis of L91, L95 colony PCR products
• Completed second part in ligating L36 and L37 by PCR
  • Digest, ligate, and transform PCR product of L36-L37 into cells

Thursday 9/2

• Primers for conversion of sigma 32 (HtpR) into RFC 10 BioBrick received
• Attempt PCR extraction, amplification and conversion to RFC 10 BioBrick of E. coli sigma 32 (HtpR)
  • Use same Colony PCR protocol as before
• Ran gel electrophoresis of sigma 32 BioBrick PCR product
  • PCR product the right size!
  • Need to assemble BioBrick construct into the correct backbone plasmid, then transform, miniprep DNA, and send out for sequencing

Friday 9/3