iGEM 2017 — Team UIOWA

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# Team:UIOWA/Notebook

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  + [Protocols](https://2017.igem.org/Team%3AUIOWA/Experiments)
  + [Medal Criteria](https://2017.igem.org/Team%3AUIOWA/Medals)
* [Human Practices](https://2017.igem.org/Team%3AUIOWA/Human_Practices)
  + [Human Practices - Silver](https://2017.igem.org/Team%3AUIOWA/HP/Silver)
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  + [Collaborations](https://2017.igem.org/Team%3AUIOWA/Collaborations)
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  + [Team](https://2017.igem.org/Team%3AUIOWA/Team)
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# Team:UIOWA/Experiments

* [Project](https://2017.igem.org/Team%3AUIOWA/Description)
  + [Description](https://2017.igem.org/Team%3AUIOWA/Description)
  + [Interlab](https://2017.igem.org/Team%3AUIOWA/InterLab)
  + [Results](https://2017.igem.org/Team%3AUIOWA/Results)
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  + [Parts](https://2017.igem.org/Team%3AUIOWA/Parts)
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* [Notebook](https://2017.igem.org/Team%3AUIOWA/Notebook)
  + [Lab book](https://2017.igem.org/Team%3AUIOWA/Notebook)
  + [Protocols](https://2017.igem.org/Team%3AUIOWA/Experiments)
  + [Medal Criteria](https://2017.igem.org/Team%3AUIOWA/Medals)
* [Human Practices](https://2017.igem.org/Team%3AUIOWA/Human_Practices)
  + [Human Practices - Silver](https://2017.igem.org/Team%3AUIOWA/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3AUIOWA/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3AUIOWA/Collaborations)
* [Team](https://2017.igem.org/Team%3AUIOWA/Team)
  + [Team](https://2017.igem.org/Team%3AUIOWA/Team)
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  + [Judging Form](https://igem.org/2017_Judging_Form?team=UIOWA)

Agarose
gels and Agarose Gel Electrophoresis

Introduction

Agarose gel electrophoresis
is useful for evaluating DNA quality, separating linear DNA fragments by size,
and for estimating relative abundance of specific DNA fragments prior to
conducting cloning procedures.

Materials

o 500 ml
dedicated flask

o Agarose
(depends on 1% agarose gel desired)

o 30mL
1X TAE buffer

o paper
towel plug

o Gel
Red (keep dark - it's light sensitive)

Procedure

* Assembling Gel
  Bed

1.
Place Gel Bed in between the Gel Bed Holder and lock in place by
pressing the Holder Walls together and locking them using the handle.

2.
Make sure the Gel Bed Holder is stable by using the Level Tool.
Adjust the Holder using the knobs until it is stable.

3.
Place Well Comb.

* Preparing the
  Agarose

4.
Add 30mL of 1x TAE Buffer to the flask.

5.
Measure and add agarose to the flask according to percentage of
gel desired.

·
1% agarose = 0.300g agarose

6.
Plug the flask with paper towel and microwave agarose and 1x TAE
buffer 1 min on High until just beginning to bubble

7.
Swirl to aid in dissolving the agarose, and continue microwaving
10 seconds at a time, until all the agarose is in solution.

8.
Discard paper plug and add 1 μl of gel red for every 10 mls of
agarose (10 μl per 100 ml).....[to be efficient use 1.5 uL in 30mL]

·
Gel Red is light sensitive so do this step fast and keep the gel
red covered.

9.
Swirl solution.

* Pour the Gel

10.   Slowly
pour into prepared gel beds (small 7 cm square) [ use 40 mls for the 10 cm
rectangular gel beds.]

11.   Remove
any bubbles (or move them away from the wells) created during pouring using a
pipet tip.

12.   Cover
the gel from light and allow to set at room temperature. (Or store Store in bags or boxes at 4oC in the dark and
create a humidified environment by adding TAE-soaked paper towels to the bag or box.)

* Gel
  Electrophoresis

13.   Remove
Well Comb and place the Gel Bed to the Electrophoresis Machine. The wells in
the gel should be closer to the Negative Terminal.

14.   Mix
10-20 uL of DNA Sample with Loading dye (5 ul per 20 uL of sample)

·
Mix by pipetting in and out or by lightly vortexing.

15.   Carefully
load the Sample DNA+Loading Dye into the well. Load 2.5 uL of DNA Ladder into a
well.

·
Keep track of what each well contains.

16.   Run @
75 V - 120 V

# Extraction of DNA fragments from an Agarose Gel

## Introduction

Enzyme contamination of DNA samples can interfere with
subsequent downstream applications. Therefore DNA fragments from enzymatic
reactions and agarose gels often must be "cleaned" or purified prior
to further use. QIAquick Kits can be used for highly efficient removal of a
broad spectrum of enzymes widely used in molecular biology. The Qiagen Gel
Extraction kit allows recovery of fragments as small as 70 bp and as large as
10 kb. Buffer QG in the QIAquick Gel Extraction Kit solubilizes the agarose gel
slice and provides the appropriate conditions for binding of DNA to the silica
membrane in the purification column. DNA adsorbs to the silica membrane in the
presence of high concentrations of salt, while contaminants pass through the
column. Impurities are efficiently washed away, and pure DNA is eluted with
Tris buffer or water. Buffer QG contains an integrated pH Indicator allowing
easy determination of the optimal pH for DNA binding. DNA adsorption requires a
pH ≤ 7.5, and the pH Indicator in the buffers will appear yellow in this range.
If the pH is >7.5, which can occur if during agarose gel electrophoresis,
the electrophoresis buffer had been used repeatedly or incorrectly prepared, or
if the buffer used in an enzymatic reaction is strongly basic and has a high
buffering capacity, the binding mixture turns orange or violet. This means that
the pH of the sample exceeds the buffering capacity of Buffer QG and DNA
adsorption will be inefficient. In these cases, the pH of the binding mixture
can easily be corrected by addition of a small volume of 3 M sodium acetate\*,
pH 5.0, before proceeding with the protocol.

## Materials

·  Buffer
QG (yellow at pH less than or equal to 7.5)

·  Buffer
PE (Add ethanol (96–100%) to Buffer PE before use (see bottle label for
volume).

·  Isopropanol
(100%)

·  a
heating block or water bath at 50°C are required.

## Procedure

* Cut the
  fragment out of an agarose gel

1.
Excise the DNA fragment from the agarose gel with a clean, sharp
scalpel on a UV box. Important: use eye and skin protection while doing this;
the UV irradiation is harmful.

2.
Weigh the gel slice in a colorless tube whose weight has been
pre-determined.

* Dissolve the
  agarose

3.
Add 3 volumes Buffer QG to 1 volume gel

·       **Note 1:** 100 mg gel ~ 100 μl.

·       **Note 2**: The maximum amount of gel per spin column is 400 mg.

·       **Note 3:** For >2% agarose gels, add 6 volumes
Buffer QG.

4.
Incubate at 50°C for 10 min or until the gel slice has
completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.

5.
After the gel slice has dissolved completely, check that the
color of the mixture is yellow (similar to Buffer QG without dissolved
agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium
acetate, pH 5.0, and mix.

* Bind the DNA
  to a Qiaquick 1column

6.
Add 1 gel volume isopropanol to the sample and mix.

7.
Place a QIAquick spin column in a provided 2 ml collection tube.
Apply the sample to the QIAquick column and centrifuge for 1 min.

8.
Discard flow-through and place the QIAquick column back into the
same tube. For sample volumes of >800 μl, load and spin again.

9.
To wash, add 750 μl Buffer PE to QIAquick column and centrifuge
for 1 min. Discard flow-through and place the QIAquick column back into the
same tube.

·       **Note 1**: If the DNA will be used for salt-sensitive applications (e.g.,
sequencing, blunt- ended ligation), let the column stand 2–5 min after addition
of Buffer PE.

10.   Centrifuge
the QIAquick column in the provided 2 ml collection tube for 1 min to remove
residual wash buffer.

·       **Note 2:** This seems trivial and is easy to forget,
but it is very important to remove all residual buffer.

* Elute the DNA
  from the column

11.   Place
QIAquick column into a clean 1.5 ml microcentrifuge tube.

12.   To
elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center
of the QIAquick membrane and centrifuge the column for 1 min.

·       **Note 1**: For increased DNA concentration, add 30 μl Buffer EB to the
center of the QIAquick membrane, let the column stand for 1 min, and then
centrifuge for 1 min.

·       **Note 2:** After the addition of Buffer EB to the QIAquick membrane,
increasing the incubation time to up to 4 min can increase the yield of
purified DNA.

* Evaluate
  Recovery

13.   Option
1: If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5
volumes of purified DNA. Mix the solution by pipetting up and down before
loading the gel. This, of course, uses up your precious sample, but is
sometimes necessary to determine whether your DNA has been recovered and
whether what was recovered is high quality.

14.   Option
2: Alternatively, use the nanospec to get a DNA concentration.

# E. coli competent cells

## Introduction

This protocol uses cold temperature incubation and commercial
(proprietary) competence buffer to maximize the number of
transformants/microgram of DNA DH5 alpha cells are a good all-purpose E. coli
strain that will be useful for our cloning projects.

## Materials

·

o Frozen stock of the
desired bacterial strain

o 2x Zymo Research Wash
Buffer

o 2x Zymo Research
Competent Buffer

o Zymo Research Dilution
Buffer

o Spectrophotometer

o Environmental shaker at
18-23oC

## Procedure

* Preparation of
  Z- Competent Cells

1.
Start 2 ml E. coli culture (HB101, DH5 alpha) in LB early in the
morning

2.
At 4-5 PM, inoculate 50 ul saturated culture into 50 ml LB in a
500 ml culture flask. Shake culture ovn (180 rpm) at 18-23 ° until the OD 600nm is
0.4 - 0.6. (18-22 hrs).

* Prepare for
  Harvesting the Cells

3.
Prepare 5 ml of 1X **Wash Buffer**by adding 2.5 ml **Dilution Buffer** to
2.5 ml of **Wash Buffer (2X Stock).**

4.
Prepare 5 ml of 1X **Competent
Buffer** by adding 2.5 ml **Dilution Buffer**
to 2.5 ml of **Competent Buffer (2X Stock)**.

5.
Store the 1X buffers on ice prior to use.

* Harvesting the
  Cells

6.
Transfer the 50 ml culture from flask to 2 50 ml cent tubes on
ice. After 10 minutes, pellet the cells by centrifugation at 4,000 rpm for 10
minutes at 0 - 4°C.

7.
Remove the supernatant and resuspend the cells gently in 5 ml
ice-cold 1X **Wash** **Buffer**.

8.
Re-pellet the cells as in Step 4.

9.
Completely remove the supernatant and gently resuspend the cells
in 5 ml ice-cold 1X **Competent Buffer.**

10.   Aliquot
(on ice) 25-100 ul of the cell suspension into sterile microcentrifuge tubes.
Cells are now ready for transformation with DNA or can be stored at -70/-80°C
for transformation at a later time.

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# Team:UIOWA/Results

* [Project](https://2017.igem.org/Team%3AUIOWA/Description)
  + [Description](https://2017.igem.org/Team%3AUIOWA/Description)
  + [Interlab](https://2017.igem.org/Team%3AUIOWA/InterLab)
  + [Results](https://2017.igem.org/Team%3AUIOWA/Results)
* [Parts](https://2017.igem.org/Team%3AUIOWA/Parts)
  + [Parts](https://2017.igem.org/Team%3AUIOWA/Parts)
  + [Basic Part](https://2017.igem.org/Team%3AUIOWA/Basic_Part)
* [Safety](https://2017.igem.org/Team%3AUIOWA/Safety)
* [Notebook](https://2017.igem.org/Team%3AUIOWA/Notebook)
  + [Lab book](https://2017.igem.org/Team%3AUIOWA/Notebook)
  + [Protocols](https://2017.igem.org/Team%3AUIOWA/Experiments)
  + [Medal Criteria](https://2017.igem.org/Team%3AUIOWA/Medals)
* [Human Practices](https://2017.igem.org/Team%3AUIOWA/Human_Practices)
  + [Human Practices - Silver](https://2017.igem.org/Team%3AUIOWA/HP/Silver)
  + [Public Engagement](https://2017.igem.org/Team%3AUIOWA/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3AUIOWA/Collaborations)
* [Team](https://2017.igem.org/Team%3AUIOWA/Team)
  + [Team](https://2017.igem.org/Team%3AUIOWA/Team)
  + [Attributions](https://2017.igem.org/Team%3AUIOWA/Attributions)
  + [Judging Form](https://igem.org/2017_Judging_Form?team=UIOWA)

# Results

![](https://static.igem.org/mediawiki/2017/1/12/T--UIOWA--3HP_ASSAY_FINAL.jpg)

![](https://static.igem.org/mediawiki/2017/6/66/T--UIOWA--3HP_FIG1_FINAL.jpg)

![](https://static.igem.org/mediawiki/2017/e/ed/T--UIOWA--3HP_FIG2_FINAL.jpg)

Our biobrick (mmsR/Pmmsa) is designed to luminesce, or emit off light, in the presence of 3HP. Induction of luminescence by 3HP is seen in Figure 2; all raw luminescence values divided by OD600 at then normalized to uninduced (0mM 3HP) values. However, induction only occurs when cells have been cultured in the presence of 3HP for 18 hours, and the growth curve in Figure 1 displays a dramatic lag in growth at 30mM and 40mM 3HP.
Further investigation is needed to optimize the performance and design of our construct.

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