Week 1
First week review
Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)
Performed a transformation in order to replicate our DNA with the constitutive GFP, and plated half of them on the plates containing chloramphenicol and used the other half for another overnight batch
Performed a mini-prep on our overnights with the constitutive GFP in order to isolate plasmids and verify that they were present through spectrophotometry
Started two separate 3A assemblies in order to piece together the pBAD promoter with the miraculin gene and the constitutive GFP gene to the SRNBC gene (homolog to Hok/Sok)
Performed a transformation in order to clone our new plasmids, one containing the pBAD promoter and miraculin gene and the other containing SRNBC and the constitutive GFP
Transformed β-cyclase, AppY, and CRTBEY genes in bacteria to grow on plates overnight
Started a new 3A assembly for the SRNBC and constitutive GFP construct along with pBAD, constitutive colicinFy, and lysostaphin
Final products were SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin
Prepared overnights for the pBAD + miraculin construct that grew on the plates and the β-cyclase, AppY, and CRTBEY genes
Performed minipreps on the overnights we had prepared the day before (for the β-cyclase, AppY, and CRTBEY genes) and shipped them off to be sequenced
Performed transformations on the SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin digests and let them grow overnight
Week 2
Miraculin
- The pBAD +miraculin construct was moved back into psb1C3 backbone and sent for sequencing
Designing gBlocks
- Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok
Week 3
Miraculin
- sequence confirmed through sequencing
- failed to extract using French press, FPLC and SDS-Page
- could be due to high arabinose induction (OD of 1)
PCR
- began work on Arduino code to cycle the machine
- purchased Peltier units and lm35 temperature sensors
Week 4
Miraculin
- induced 3 test cultures with 0%, 0.05%, 0.1%, 0.2% arabinose with no significant results
- will perform procedure again using bl21 instead of Dh5a
PCR
- received Peltier's, lm35's and began testing the code
- 6 volt battery pack was found to be insufficient to run Peltier's
- purchased an AC/DC power converter
Week 5
Miraculin
- transformation in BL21 strain was successful
- SDS page showed a band at roughly 25 kDa
Hok/Sok
- inserted Hok/Sok gblock into PSB1C3 backbone using Gibson assembly
- construct sent for sequencing
Interlab Study
- transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP
PCR
- Peltier units heated and cooled to proper temperatures however took 30 minutes to cycle properly
- started construction of housing to insulate the element to cycle temperature faster and reduced time to 15 minutes
- burned out Peltier elements and broke temperature sensor, and ordered new ones
Week 6
Hok/Sok
- transformed unstable GFP and RFP with PBAD into dh5a
- transformed unstable RFP and inducible lac promoter (k1399011) and const_promoter+RBS (K081005) into dh5a
- ligated const_promoter + RBS + RFP
Interlab
- performed a 3A assembly of each promoter + GFP in PSB1K3 (GFP in PSB1A3 and promoters in PSB1C3)
PCR
- received new Peltier unit and temperature sensor
- began milling on metal PCR tube holder
Week 7
Hok/Sok
- construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing
- Sequencing showed a 3A assembly of quick degrading RFP and a "leaky" lactose promoter
- Re- ran 3A assembly but the transformation failed
- could be due to contaminated SOC media
PCR
- received milled PCR tube holder
- embedded temperature sensors into holder reran the machine and again burned out Peltier's
- spoke with Peltier manufacturers and Open PCR staff and found the Peltier's used for PCR were specialized and more expensive
- bought higher grade Peltier's to handle high voltages
Week 8
Hok/Sok
- 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
- 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
- ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene
PCR
- Peltier's were still in shipping however work continued on the software
- designed relay configuration to both heat and cool Peltier's with an unidirectional current flow
Week 9
Hok/Sok
- 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
- 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
- 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
Week 10
Hok-Sok
- created construct with RFP and hok/sok
- Pcr of the construct failed so the construct will be sequenced to check
PCR
- cycle data was taken and it exhibits consistency
Week 11
Hok/Sok
- 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
- 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
- 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
Week 12
Hok/Sok
- Ran a Gibson Assembly of Hok/Sok + RFP
Interlab
- worked on finishing the last construct
- K13 sequenced correctly and the pcr looks good
- contacted W&M on possible collaboration for the last construct
Lutein
- ordered primers for Gibson Assembly
PCR
- replaced old relays with higher powered relays
- reduced mass by over 50% of the peltier machine
- resulted in speedier temperature changes
Week 13
Hok/Sok
- 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
- 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
- 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
- increased the denaturation temperature, attained an amplified product
Week 14
Hok/Sok
- 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
- obtained the last construct from W&M
- tested the last construct using the plate reader
- Turned in the IL study
PCR
-began work on incubator
-incubator was proven to maintain 37 degrees Celsius
Week 15
Hok/Sok
- Tested generations alpha beta and gamma in BL21 cells
- Alpha and beta contained groups A-E
- gamma had only group F
Week 16
Hok/Sok
- Tested generation delta using DH5alpha
- contained groups A-E
