iGEM 2017 — Team U_of_Guelph

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# Team:U of Guelph/Notebook

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# Notebook

![](https://static.igem.org/mediawiki/2017/4/4a/T--U_of_Guelph--gryphon.jpg)

# Experiment Overview

Here we will include a day by day entry for our project. We need to make sure we atleast include the days we did which experiments but more information would be good too. Note who participated in what should be included.

# June 5

LB and LB Kan Plates were poured.

# June 6

Re-streaked Ecoli BL21, DH5α, and DH5α/ pET-28a from the stocks we were provided.

# June 7

Liquid cultures of DH5α, DH5α/ pET-28a and BL21 were inoculated and grown for 48 hours

# June 9

Glycerol stocks were prepared from the liquid cultures

# June 14

Ordered *frc* and *oxc* Full length Sequences

# June 19

Ordered gBlock fragments of *frc* and *oxc* for use in a gibson assembly

# June 20

Ordered original PCR primers to add PstI to pET-28a

# June 21

A half sleeve of each chloramphenicol and Kanamycin plates were poured, DH5α/pET-28a was re-streaked, and a DH5α liquid overnight culture was prepared.

# June 22

DH5α competent cells were made. Experiment failed at the final step due to forgetting the glycerol before putting cells in liquid nitrogen.

# June 25

Ordered primers to add *frc* and *oxc* ends to pET-28a(PstI).

# July 5

Miniprep DH5α/pET-28a, and PCR to add PstI to pET-28a.

# July 6

Gibson Assembly transformation.

# July 10

Made LB broth and an agarose gel.

# July 12

Re Streaked BL21 (onto LB) and DH5α/pET-28a (onto LB Kan).

# July 13

Overnight liquid cultures were prepared of BL21 and DH5α / pET-28a in 4 mL LB broth in plastic culture tubes. Kanamycin was forgotten in pET-28a culture. Ordered Primers for *frc* and *oxc* genes without RE end sites.

# July 14

Prepared BL21 Competent Cells, Miniprep of DH5α / pET-28a.

# July 19

Inoculation of DH5α/pET-28a overnight liquid culture incubated at 37 degrees with shaking.

# July 20

Miniprep of DH5α/pET-28a liquid culture.

# July 21

PCR

# July 24

PCR of pET-28a to add PstI RE site using incorrect Primers

# July 28

PCR of pET-28a to add PstI RE site using incorrect Primers

# July 29

Gel of *frc* and *oxc* PCR

# August 3

pET-28a (PstI) PCR

# August 8

Ordered New PstI Primers

# August 10

PCR of pET-28a to add PstI cut site using new primers

# August 15

Agarose Gel

# August 17

Ordered New *frc* and *oxc* Primers

# August 19

Transformation of pET-pstI (66.9 and 68.9) into DH5α. Incubated plates all weekend.

# August 21

Re Streaked colonies 1-10 from pET-28a (PstI) transformation plates.

# August 22

Final PCR of *frc* (47.0, 47.7, 49.1 and 51.2) and *oxc* at (53.8, 55.8, 57.1, 58.0)
Liquid cultures of colonies 1-6 from plates. Purification of *frc* and *oxc* PCR products.

# August 23

Gel of *frc* and *oxc* PCR reactions. Miniprep of DH5α pET-28a(Pst1). RE digest pET-28a(Pst1) with PstI and AnaI. Gel of RE digest DH5α pET-28a(Pst1) with PstI and AnaI. RE digest pET-28a(Pst1)(2) EcoRI and PstI, and *frc* (47.0 and 49.1) and *oxc* (53.8 and 55.8). Ligation of pET-28a(Pst1)(2) with *frc* and *oxc*. Transformation of DH5α with ligation mixtures, +ve control pET-28a(PstI) (2) and -ve control RE digested pET-28a(PstI) (2)

# August 24

Gel of ligation mixtures after transformation failed
Transformation of DH5α with ligation mixtures, +ve control pET-28a(PstI) (2) and -ve control RE digested pET-28a(PstI) (2)
Plated on Kan plates and incubated overnight at 37 degrees.

# August 25

Put plates in fridge to store until monday.

# August 28

Re-streaked pET-28a(PstI) (2) to obtain isolated colonies. Suspended selected colonies in 10 ul of water. Colony PCR and agarose gel. Liquid overnight cultures of DH5α/pET-28a(PstI)*frc* (colony 14 and 17) and DH5α/pET-28a(PstI)*oxc* (colonies 1, 3 and 5)

# August 29

Re-streaked pET-28a(PstI) (2) to obtain isolated colonies. Made new sterile LB. Minipreped DH5α/pET-28a(PstI)*frc* (colony 14 and 17) and DH5α/pET-28a(PstI)*oxc* (colonies 1, 3 and 5). RE digested DH5α/pET-28a(PstI)*frc* (colony 14 and 17) and DH5α/pET-28a(PstI)*oxc* (colonies 1, 3 and 5) with EcoRI and PstI. Made glycerol stocks of DH5α/pET-28a(PstI)*frc* (colony 14 and 17) and DH5α/pET-28a(PstI)*oxc* (colonies 1, 3 and 5) and stored in Tetlow lab -80 freezer

# August 30

Dry autoclave run to sterilize tips and tubes. RE digested PSB1C3 with EcoRI and PstI. Ligated *frc* (47.0 and 49.1) and *oxc* (53.8 and 55.8) into PSB1C3. Transformed PSB1C3frc and PSB1C3oxc into DH5α and plated on Kan plates, +ve control pET-28a (PstI), -ve control RE digested PSB1C3. Liquid culture of pET-28a(PstI) (2)

# August 31

Realized PSB1C3 carries Chloramphenicol resistance and transformations need to be redone.

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University of Guelph iGEM 2017


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# Team:U of Guelph/Experiments

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[Notebook](https://2017.igem.org/Team%3AU_of_Guelph/Notebook)
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[Attributions](https://2017.igem.org/Team%3AU_of_Guelph/Attributions)

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# Experiments

![](https://static.igem.org/mediawiki/2017/4/4a/T--U_of_Guelph--gryphon.jpg)

# Experiment Overview

Our experimental goal this year was to clone *frc* and *oxc* into *E. coli* DH5α using pET-28a. To accomplish this goal we had *frc* and *oxc* synthesized by IDT and used PCR to add the correct restriction enzyme sites. Site directed mutagenesis was used to add the PstI cut site to pET-28a, then *frc* and *oxc* were inserted using standard restriction enzyme digests and ligation. pET-28a*frc* and pET-28a*oxc* were then transformed into DH5α.

# Preparation of Competent Cells

This year we prepared two different types of competent cells, DH5α and BL21. We used a chemical competency method involving calcium chloride. As the final step in preparation all cells were frozen in either 50 µl or 100 µl volumes by submersion in liquid nitrogen. Competent cells were then stored in a -80°C freezer for further use.

# pET-28a isolation

pET-28a was purified from DH5α/pET-28a cells provided by Cezar Khursigara's Lab using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20°C freezer for further use.

# Site Directed Mutagenesis of pET-28a

PCR was used to conduct site directed mutagenesis to add a PstI cut site to pET-28a. Two basepairs were changed at the wild type XhoI cut site to change the sequence from ctcgag to ctgcag. Primers were ordered from IDT.

# Confirmation of SDM Success

A double restriction enzyme digest was conducted to ensure that the PstI cut site had been added correctly. A single restriction enzyme digest and undigested plasmid were used as controls. All digestion products were run on an agarose gel and visualized using either Novel Juice (FroggaBio) or SyBr. Samples were frozen in a -20°C freezer for further use.

# Isolation of pET-28a(PstI) from DH5a

pET-28a was purified from DH5α/pET-28a cells using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20°C freezer for further use.

# Addition of EcoRI and PstI to *frc* and *oxc*

PCR was used to add EcoRI and PstI cut sites to the ends of *frc* and *oxc*. PCR primers and synthesized *frc* and *oxc* were all obtained from IDT.

# Adding *frc* and *oxc* to pET-28a

The genes *frc* and *oxc* were added individually to pET-28a using standard ligation techniques.

# Transformation of pET-28a*frc* and pET-28a*oxc* into DH5α

Using standard heat shock techniques pET-28a*frc* and pET-28a*oxc* were transformed into DH5α.
Transformed cells were incubated for 1 hour at 37°C with rotation (using our homemade rotator) then plated on Kanamycin plates.

# Confirmation of the Presence of *frc* and *oxc*

Colony PCR was used to confirm the presence of *frc* and *oxc* from the colonies grown from the transformation. One of each type of colony was selected to make liquid culture, and then glycerol stocks from for long term storage of the transformed bacteria.

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University of Guelph iGEM 2017

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# Team:U of Guelph/Results

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Project

[Description](https://2017.igem.org/Team%3AU_of_Guelph/Description)
[Design](https://2017.igem.org/Team%3AU_of_Guelph/Design)
[Experiments](https://2017.igem.org/Team%3AU_of_Guelph/Experiments)
[Notebook](https://2017.igem.org/Team%3AU_of_Guelph/Notebook)
[Results](https://2017.igem.org/Team%3AU_of_Guelph/Results)
[Attributions](https://2017.igem.org/Team%3AU_of_Guelph/Attributions)

[Judging Form](https://igem.org/2017_Judging_Form?team=U_of_Guelph)

# Results

![](https://static.igem.org/mediawiki/2017/4/4a/T--U_of_Guelph--gryphon.jpg)

# Results

This year *frc* and *oxc* were cloned into DH5α/pET-28a.

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University of Guelph iGEM 2017

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