Team:UMaryland/Notebook - 2015.igem.org

Miraculin:
ligated the pBAD promoter in PSB1C3 with the miraculin gene in PSB1C3 using 3A assembly
plate had colonies

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

Week3

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 /Gibson Check
performed a series of check in order to ensure that all the reagents are active
everything seems to be working
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