iGEM 2015 — Team Concordia

# Team:Concordia/Notebook

* [![](https://static.igem.org/mediawiki/2015/4/48/Concordia2015_iGEM-logo.png)](https://2015.igem.org/Team%3AConcordia)
* OUR TEAM
  [+ Team Members](https://2015.igem.org/Team%3AConcordia/Team)
  [+ Attributions](https://2015.igem.org/Team%3AConcordia/Attributions)
  [+ Contact Us](https://2015.igem.org/Team%3AConcordia/Contact)
* PROJECT
  [+ Description](https://2015.igem.org/Team%3AConcordia/Description)
  [+ Lactococcus Lactis](https://2015.igem.org/Team%3AConcordia/Lactococcus_Lactis)
  [+ Scaffold](https://2015.igem.org/Team%3AConcordia/Scaffold)
  [+ Food Intolerances](https://2015.igem.org/Team%3AConcordia/FoodIntolerance)
  [+ Prototype Pathway](https://2015.igem.org/Team%3AConcordia/Pathway)
* RESULTS
  [+ Results of Cloning](https://2015.igem.org/Team%3AConcordia/ResultsCloning)
  [+ Results of Testing](https://2015.igem.org/Team%3AConcordia/Results)
  [+ Future Work](https://2015.igem.org/Team%3AConcordia/FutureWork)
* WET LAB
  [+ Notebook](https://2015.igem.org/Team%3AConcordia/Notebook)
* PARTS
  [+ Team Parts](https://2015.igem.org/Team%3AConcordia/Parts)
* POLICIES
  [+ Human Practices](/Team%3AConcordia/Practices)
  [+ Outreach](https://2015.igem.org/Team%3AConcordia/Outreach)
  [+ Collaborations](https://2015.igem.org/Team%3AConcordia/Collaborations)
  [+ Safety](https://2015.igem.org/Team%3AConcordia/Safety)

## Notebook

#### Week 1

**Wet lab work:**

Tuesday:
-Initial PCR for coh2S1 and CWAm6 parts from pAW576
-Purification and digestion of the parts and the backbone with EcoRI and PstI

Wednesday:
-Initial PCR for PnisA and SPusp45 from pAW576 and IDT G-Blocks, only PnisA was amplified
-Initial PCR for CBD and coh2S1 from ordered IDT G-Blocks (group 2), not successful

Thursday:
-Ligation and transformation attempt of the plasmids containing coh2S1 and CWAm6 following the iGEM protocol, but with small concentrations and no results
-Second attempt at amplifying the G-Block parts with different primers, no success
-Digest of amplified and purified PnisA with EcoRI and PstI and of the backbone

Friday:
-Blunt-end cloning to amplify the G-Block parts from a pJET vector
-PCR amplification, digest, ligation and transformation of coh2S1 and CWAm6, no results

Saturday:
-Second attempt at the same ligation using a new digested backbone and a new T4 ligase

Sunday:
-No results from Saturday and probably some contamination on the plates.

**Dry lab work:**

Monday:
-Research on our research subject and our project
-Preparation of a presentation to our supervisors and professors about our project organization and timeline

Tuesday:
-Presentation in front of our supervisors and professors

Wednesday:
-Sponsorship package

Thursday:
-Sponsorship package

Friday:
-Restriction enzymes order received

Saturday:
-Sponsorship package
-Talk about fundraising ideas

Sunday:
-Sponsorship package
-First thoughts on a bake sale soon

#### Week 2

**Wet lab work:**

Our team has now worked in the lab for two weeks straight with some results and a lot of troubleshooting. The first part of our scaffold is finally ready for sequencing and maybe submission. Other parts are on their way and may be ready any day by now. A second round of primers have been ordered and will be used to amplify the last components of our scaffold. The iGEM compatible expression vector now has its final design and its components will be ordered soon. Also, we started working on our fusion enzymes by amplifying their docking domain; while the enzymes themselves are still being discussed. α-galactosidase is the latest idea for our digestive probiotic bacteria !

**Dry lab work:**

We started a project on gofundme.com in order to raise money and send the whole team to the Boston Jamboree, and our sponsorship package will be reviewed and send in the next few days. We finally presented our entire project organization to our supervisors and are currently looking for teams to collaborate with. For this purpose, we contacted the other Quebec team, iGEM Sherbrooke, to organize a regional meeting. We could also extend to others teams by attaching their enzymes on our scaffold. Finally, we will officially be at the international startup festival hosted in Montreal Saturday the 18th of July to make demonstrations on Biology and talk about our project.

#### Week 3

**Wet lab work:**

Work finally paid off this week in the iGEM Concordia lab. Our troublesome GBlocks parts are turning around and after troubleshooting for bad antibiotics in our plates we succeeded in inserting XDock2, CBD, coh2S1 and CWAm6 into their backbone, making it five parts ready to be submitted to iGEM. Our first sequencing order also confirmed the insertion of our promoter from last week. Assembly of the scaffold construct can therefore start and the last missing parts will be ready soon. Concerning our iGEM compatible expression vector, the first part is now in its backbone and we only need the MCS from IDT to be ordered and inserted before we can use it.

**Dry lab work:**

On the dry lab aspect of our project, our sponsorship package has been modified and reviewed all week and a final version is to be expected soon. A samosa and cupcake sale was organized during the week in the university and in the streets of Montreal in order to raise money to go to Boston. Also, our intervention at the Montreal International Startup Festival under the Bricobio booth was a success! Kids loved our strawberry DNA extraction and we had a chance to reach out of the community and explain our project. Finally, we are still looking for iGEM teams ready to collaborate with us, especially if they have are working on an application for our extracellular scaffold or our iGEM compatible expression vector.

#### Week 4

**Wet lab work:**

The work on our parts is going super tight for the wet lab team. The terminator of our scaffold construct has been added to the list of our parts ready to be sequenced. We're now going to start assembling parts together. We also started working on the chromoproteins we'll use to test the scaffold, and the first component of our expression vector is ready. A new order for IDT primers and GBlocks was sent during the week for the next steps of our project. Finally, we are currently discussing how to test and characterize our parts to present our results.

**Dry lab work:**

A lot has been done on the dry lab side too. Our sponsorship package is finally complete and will be sent to potential sponsors within a few days. We also started doing reading on the different aspects of our project to get ready for our final presentation.

#### Week 5

**Wet lab work:**

Only one of our parts is not ready for submission , but should will be ready soon. We started putting the scaffold components together this week. With the latest IDT order having arrived, we are now able to prepare our test enzymes as well as our Biobrick expression vector.
As some of us continue getting the constructs ready, the rest of the team is going to train on manipulating Lactococcus lactis and on protein purification. We are currently also discussing what to do with the scaffolds and the best ways to characterize them.

**Dry lab work:**

Our sponsorship package has finally been sent to companies who can potentially help us develop this project. The common design for our wiki, poster and presentation is starting to be discussed and we're hoping to see its first drafts soon. We are still looking for other iGEM teams who would want to collaborate, possibly testing our two projects together.

#### Week 6 & 7

**Wet lab work:**

Our main construct is only a few cloning rounds away now ! Our efforts are focused on both the scaffold and the enzymes fusions as we decided to test six different enzymes to illustrate three proofs of concept for our project. The iGEM compatible expression vector that we designed had also been constructed and will help us express and purify our enzyme fusions. We will soon start working with our model organism Lactococcus Lactis and manipulating proteins. Although we have discussed testing of our constructs, some precisions are being explored as how to assay and measure specific enzymatic activities.

**Dry lab work:**

The first designs that will be presented in Boston have been created as well as a logo for the 2015 team. Work on the wiki is also increased to get design and text ready to be uploaded. We are currently trying to organize a talk at our university orientation week to introduce new students to synthetic biology, which would be a great occasion to start forming a team for iGEM 2016!

#### Week 8

**1. Lab-based research:**

Monday:
-PCR cleanup of digested LacZ
-ligation/transformation of pICEL (failed)
-digest of pICES1, pET15BB, gel and extraction
-Ligation/transformation of pETSD1 in BL21DE3 (confirmed)
-Inoculations: chromoproteins in pJET and Docks
-order primers for ALD2 and ADH2
-got yeast genomic DNA from Damien
-digest of SacC with Eco/Age

Tuesday:
-Colony PCR of chromoproteins
-other colony PCR checks
-gel of digests
-Inoculations
-minipreps
-Power failure (thank you Concordia)
-gel and extract SacC digested
-ligation/transformation of pICES2 (failed)

Wednesday:
-Minipreps
-check digest + gel
-Colony PCR + gel (for Kevin)
-Digest and gel extraction of the two dockerins
-Ligation/ transformation of aeblue + eforred with docks
-Power failure, episode 2
-Looked at sequencing data
-Inoculations
-confirm pETSD1 from former colony PCR and digest (given to Adithi for purification)
-wrote “chromoprotein proof of concept” text for the wiki

Thursday:
-order aeBlue and eforRed chromoproteins as GBlocks (troubleshooting)
-gel extract LacZ PCR
-digest LacZ (Xba/Pst) and pSB1C3 (Xba/Pst), cleanup and gel extraction
-gel of pICL22 digest (for Kevin)
-ligation/transformation of pICEL, pICES2 (worked)
-PCR ALD2 and ADH2 twice (no primer dimers and 1 single small band, temp gradient)
-discuss troubleshooting with Lauren and Adithi
-new PCR of LacZ (failed)

Friday:
day off, Adithi and Carlo made colony PCR to confirm pICES2 and inoculation

Saturday:
-LB+Cm plates stock
-miniprep pICEL, pICES2 and digest, gel and extraction
-ligation/transformation of pICES2 (backup) and pICEL1, L2 (failed)
-Inoculate pICE01, 02, S2 for digest
-Ordered ADH2 and ALD2 and new Rprimer from IDT

Sunday:
-Colony PCR pICEL1, L2, S2 (failed because of badly digested backbone)
-PCR mastermix made
-made Phosphate buffer (50mM, 300mM NaCl, pH 5.98) for cell washing
-Miniprep (Sam) and check digest (confirm pICES2)
-digest of pICES2, pSB1C3 (Xba/Pst), gel (digest failed)
-re-digest set up overnight
-Inoculation of pICEL, pICES2 (Adithi)
-Plate pICL31 transformation (for Kevin)
-Order NgoMIV

**2.Dry lab work:**

texts written: chromoproteins proof of concept, parts description,
ideas on new logo, poster, wiki organization,
talked to Sam about Beyond the bench part

**3.Training:**

3.1. Lab methods and bioinformatic tools learned:
phosphate buffer,
PCR from genomic DNA,
Inducing expression,
Lactis growth

**4. Assessment of progress/productivity**

4.1 Estimated hours working on iGEM project (reason for low hours if applicable):
a lot
4.2 Approximate % carry-over from last meeting (indicated above with \*):
50
4.3 Rate your progress (1-10):
slow but steady (4)
4.4 Upcoming deadlines:
Get all the proteins ready to be purified asap