Difference between revisions of "Team:UMaryland/Notebook"

Revision as of 01:47, 19 September 2015

Notebook

Week 2
Week 4
Week 6
Week 8
Week 10
Week 12
Week 14
Week 16

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
Miniprep
analyzed sequences
prepared 3A assembly clone of pBAD-miraculin

3A assembly
two backbones
pSB1C3
pBAD promoter + genes for miraculin
cut out pBAD with 2 enzymes (EcoRI and SpeI) and cut out miraculin (XbaI and PstI)
want to put pBAD in front of miraculin
put into backbone w/ different antibiotic resistance than original backbone: pSB1A3 (means of selection)
plate had colonies
Human Practices
Kevin got in contact w/ Bioacademy program @ Northwest High School Trying to set up a talk for this Wednesday Kevin + Adam + Dylan + Juhye going goal to target various age groups create game fundamentals of genetics via presentations, arts, etc. Conference scheduled for June 25th 4 teams confirmed: UVA, UMBC, Stonybrook, Duke contact iGEM headquarters to inform them about meetup → Robert
Kevin got in contact w/ Bioacademy program @ Northwest High School

Trying to set up a talk for this wednesday

Kevin + Adam + Dylan + Juhye going

goal to target various age groups

create game

fundamentals of genetics via presentations, arts, etc.

Conference scheduled for June 25th

4 teams confirmed: UVA, UMBC, Stonybrook, Duke

contact iGEM headquarters to inform them about meetup → Robert

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

@@ Line 259: / Line 259: @@
 First week review
 <ul>
-<li>Cleaned the lab</li>
+<li>Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)</li>
-<li>moved in iGEM materials</li>
+<li>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</li>
 <li>Miniprep<li>
 <li>analyzed sequences</li>