- const_promoter + RBS + RFP were replated from last week but produced no colonies
- re-transformed original and new 3A assembly which produced the correct sequence
- ligated const_promoter:QD-RFP in PSB1C3
- performed a 3A assembly of Hok/Sok plasmid with QD-RFP downstream in PSB1K3
- site directed mutagenesis of quick degrading GFP (Bba_K750000) failed
- there was no change from the original sequence
Interlab study
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
3A assembly attempts for the promoters and GFP failed
Gibson Assemblies produced a vast amount of colonies
questionable success because the amount of colonies may indicate false positives or contamination
Lutein
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene
PCR
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
previous 3A assembly from last week showed no colony growth on kanamycin plate
transformed RFP into C3 backbone last week
colonies were produced that were not distinctively red but with the correct sequence
3A assembly with the RFP + Hok/Sok failed
Created primers for Gibson assembly to create Hok/Sok +const_promoter::RBS::unstable RFP
Interlab Study
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful
K08 and K13 constructs grew colonies
Miniprepped constructs and only promoter K08+GFP had the correct sequence
PCR
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
received high rated peltier units that did not break
took ~31 minutes to complete 1 cycle but insulated heating dropped the time to 16 minutes
Took apart a hair dryer for heating element
heating went to >95 in ~2 seconds, cooled to 50 in ~8 seconds
The heating element is not very precise so it resulted in a lot of overshoot in temperature
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
3A assembly of H/S + RFP failed
ran a PCR of PSB1C3+H/S+RFP using 3 different rxn buffers
HF rxn buffer
GC rxn buffer
GC rxn buffer w/ DMSO which yielded product
Ran a PCR of unstable RFP using the 3 rxn buffers above
Interlab Study
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
used Phusion instead of Q5 for PCR
ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products
Gibson Assembly of GFP + IL1 produced colonies
PSB1C3 - IL3 did not have bands in gel of appropriate size
Lutein
RE digests of pLAC-RFP in PSB1C3
ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies
PCR Machine
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
rebuilt top of hair dryer housing w/ soda can
observed random temp spikes occurring during each run
caused by hardware issue with relays
purchased new relays to test this week
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
sequence of hok/sok construct created last week was incorrect
may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson
Interlab
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
Gibsons of IL3 (K13 + GFP) have all failed
3 trials of IL2 , IL1,+ control (const_promoter with GFP), - control were tested using the plate reader
IL2 was a little low in fluorescence, similar to the - control
PCR
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
Rewired hair dryer and changed relays to handle higher wattage
attempted PCR cycle failed
assumed temp sensor needed to be immersed in mineral oil to properly report temp
denaturation temp may have been too low
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system
Group A: RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
Group B: RFP coding region; constitutive promoter ; no chloramphenicol in liquid culture
Group C: RFP coding region; no promoter ; chloramphenicol in liquid culture
Group D: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
Group E: H/S-RFP coding region; constitutive promoter ; nochloramphenicol in liquid culture
Group F: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
Interlab:
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
Obtained the last construct from W&M
Tested the last construct using the plate reader
Turned in the IL study
