iGEM 2015 — Team UMaryland

# Team:UMaryland/Notebook

* [Information](https://2015.igem.org/Team%3AUMaryland/about)
  + [About](https://2015.igem.org/Team%3AUMaryland/about)
  + [Our Team](https://2015.igem.org/Team%3AUMaryland/Team)
  + [Acknowled- gements](https://2015.igem.org/Team%3AUMaryland/Attributions)
  + [Fundraising](https://2015.igem.org/Team%3AUMaryland/fundraising)
  + [2015 Jamboree](https://2015.igem.org/Team%3AUMaryland/After)
* [Project](https://2015.igem.org/Team%3AUMaryland/Description)
  + [Description](https://2015.igem.org/Team%3AUMaryland/Description)
  + [Hok/Sok](https://2015.igem.org/Team%3AUMaryland/HokSok)
  + [PCR Design](https://2015.igem.org/Team%3AUMaryland/Design)
  + [Results](https://2015.igem.org/Team%3AUMaryland/Results)
  + [Side Projects](https://2015.igem.org/Team%3AUMaryland/sideprojects)
  + [Interlab Study](https://2015.igem.org/Team%3AUMaryland/Interlab)
* [Parts](https://2015.igem.org/Team%3AUMaryland/Part_Collection)
  + [All Parts](https://2015.igem.org/Team%3AUMaryland/Part_Collection)
  + [Basic Parts](https://2015.igem.org/Team%3AUMaryland/Basic_Part)
  + [Composite Parts](https://2015.igem.org/Team%3AUMaryland/Composite_Part)
* [Outreach](https://2015.igem.org/Team%3AUMaryland/Practices)
  + [Human Practices](https://2015.igem.org/Team%3AUMaryland/Practices)
  + [Regional Meetup](https://2015.igem.org/Team%3AUMaryland/Collaborations)
  + [Survey](https://2015.igem.org/Team%3AUMaryland/Survey)
* [Notebook](https://2015.igem.org/Team%3AUMaryland/Notebook)
  + [Journal](https://2015.igem.org/Team%3AUMaryland/Notebook)
  + [Protocols](https://2015.igem.org/Team%3AUMaryland/protocols)
  + [Safety](https://2015.igem.org/Team%3AUMaryland/Safety)
* [iGEM Main](https://2015.igem.org/Main_Page)

[![Home](https://static.igem.org/mediawiki/2015/3/3d/Umd-igem2.png)](https://2015.igem.org/Team%3AUMaryland)

**Notebook**

* [Week 2](#Week2)
* [Week 4](#Week4)
* [Week 6](#Week6)
* [Week 8](#Week8)
* [Week 10](#Week10)
* [Week 12](#Week12)
* [Week 14](#Week14)
* [Week 16](#Week16)

**Week 1**

Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes

* - 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
    * - 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 (for lutein project) 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

* - RE digest on β-cyclase, AppY, CREB, and CRTBEY genes along with the pSB1C3 backbone and the pBAD + miraculin out of the pSB1A3 backbone so that we could move the genes to the pSB1C3 backbone
* - Ran gel with all parts and cut out the bands
* - Mini-prepped SRNBC + constitutive colicinFy and shipped them off to be sequenced
* - Performed gel purification on gels from day before but they failed; they didn’t show up in the spectrophotometer
* - Performed minipreps on pBAD+Miraculin in the pSB1C3 backbone as well as the const. GFP+SRNBC
* - Performed RE digests on the previous mini-preps: EcoRI and PstI on pBAD + Miraculin, and XBa1 and Pst1 on SRNBC + Constitutive GFP

Designing gBlocks

* - Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok, and ordered them

**Week 3**

Miraculin

* - Sequence of pBAD + Miraculin confirmed through sequencing
* - Culture of pBAD + Miraculin was induced with 0.1% arabinose at OD of 1
* - Attempted to purify the Miraculin out of the induction culture by first using a French Press to lyse the cells and then running it through cobalt bead column by FPLC (Fast Protein Liquid Chromatography)
* - Ran the resulting 62 elutions through an SDS-PAGE
* - Failed to extract Miraculin using French press, FPLC and SDS-Page
* - Made overnights of pBAD + Miraculin culture to prepare for test inductions

SRNBC and Hok/Sok

* - Performed a gel extraction of SRNBC, const. GFP, and pBAD which we then ligated them together to make the SRNBC construct
* - Performed transformations of the constitutive GFP + SRNBC construct
* - Transformations of SRNBC + Constitutive GFP failed
* - Performed PCR on the pSB1C3 backbone in order to amplify it in preparation for a Gibson Assembly with the Hok/Sok gene

PCR

* - Began work on Arduino code to cycle the machine
* - Purchased Peltier units and lm35 temperature sensors

**Week 4**

Miraculin

* - Made overnights of pBAD+Miraculin culture to prepare for test inductions tomorrow
* - Inoculated 3 test cultures of pBAD+Miraculin in LB broth with Ampicillin
* - Induced the tubes with 0.05%, 0.1%, and 0.2% arabinose in order to initiate production of miraculin at an OD of 0.4
* - Prepared the induction samples for SDS-PAGE by separating out the media, supernatant after lysing with 200 uL SDS and then the supernatant after lysing with SDS again (pellet supernatant)
* - Ran samples of 0%, 0.05%, 0.1%, and 0.2% arabinose through the gel, but no bands appeared
* - Prepared to perform procedure again using bl21 cells instead of Dh5a cells

Hok/Sok

* - Performed an RE digest on the pSB1C3 backbone with XbaI and SpeI and then ran it through a gel to separate it out, to prepare for the moving of Hok/Sok into pSB1C3

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