Team:UMaryland/Notebook - 2015.igem.org

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)

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

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

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)

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

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

- code made and proven to properly cycle machine

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 machine

- 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 Machine

- 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

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

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