Team:OU Norman/Project/Protocols

From 2014.igem.org

Antibiotic (Tetracyclineb) Stock

Stock Solution

Concentration	Storage
5 mg/mL in EtOh	-20°C

Working Concentrations

Stringent Plasmids	10 μg/mL
Relaxed Plasmids	50 μg/mL

Physical Data Table

Anitbiotic	Molecular Weight	Mode of Action
Tetracycline Hydrochloride	480.9 g/mol	Inhibits bacterial protein synthesis Blocks ribosomal binding of aminoacetal-Trna

PROCEDURE FOR PREPARATION

Obtain (3) 15 mL test tubes
Obtain (1) 50 mL test tube
Obtain 0.250 g of tetracycline

Place in the 50 mL test tube

Add EtOH to the 50 mL mark

Shake to dissolve completely
Use 100% EtOH

Because the tetracycline did not dissolve, we removed 15 mL of the solution and added 15 mL of water to catalyze the reaction

Next time we will use 35 mL of EtOH and 15 mL of water

Pour 15 mL of the solution into each of the 15 mL test tubes

Mark the test tubes in blue to identify tetracycline

PROCEDURE FOR BROTH AND PLATES

Add 500 μL of the tetracycline antibiotic twice to the broth using an Eppendorf pipette

Make sure the broth is cool before adding the antibiotic

Pour the broth into 25 plates

Allow the plates to cool before stacking them

While the plates are cooling, place a second broth container on the spin plate

Temperature = 150°C
Rotation = 1000

Once the plates have cooled mark them in blue (to identify as tetracycline), put them back in the sleeve, and store in the 5th floor refrigerator

Ligation of ClosOri, m1sR, and psB1K3

PROCEDURE

Obtain (2) 1.5 mL centrifuge tubes
In one of the centrifuge tubes, add:

20 μL of heat killed digest of ClosOri
20 μL of heat killed digest of psB1K3
1 μL of T4 ligase
5 μL of 10x T4 ligase buffer
4 μL of nuclease free PCR H2O

In the second centrifuge tube, add:

20 μL of heat killed digest of m1sR
20 μL of heat killed digest of psB1K3
1 μL of T4 ligase
5 μL of 10x T4 ligase buffer
4 μL of nuclease free PCR H2O

Restriction Digest of ClosOri and m1sR

Conversions
ClosOri: 500 ng x 1 μL/ 210.2 ng = 2.38 μL
m1sR: 500 ng x 1 μL/ 470.6 ng = 1.05 μL
Backbone (psB1K3): 500 ng x 1 μL/ 175.2 ng = 2.86 μL
Physical Data Table

	EcoRI	SpeI	10x Buffer	DNA	PCR H2O	Total
ClosOri	2μL	2μL	2μL	2.38μL	11.62μL	20μL
m1sR	2μL	2μL	2μL	1.06μL	12.94μL	20μL
psB1K3	2μL	2μL	2μL	2.86μL	11.14μL	20μL
psB1K3	2μL	2μL	2μL	2.86μL	11.14μL

PROCEDURE

Cross link water for 30 seconds

Use DI water
Take cap off of DI water when in spectrolinker

Add appropriate amount of water for each of the four centrifuges (1.5 mL)

Remember to keep the caps of the centrifuges on until they are in action
Stick the pipette all the way down to the bottom of the centrifuge to ensure complete utilization of water

Add buffer
Add appropriate amounts of DNA
Add restriction enzymes
Spin the samples to briefly collect all of the mixture
Incubate the restriction digests at 80°C for 15 minutes

Use a thermal cycler with a heated lid

Cool on the bench for ~5 minutes

Rescue of BBa K542003

DNA Sample = 2M on plate 1
Plasmid = pSC1C3

pBAD Regulated TetR
Production of TetR is regulated by the pBAD promoter. TetR is an inhibitor of the constitutively “on” promoter pTet-BBa_R0040. Therefore, this part may be used in conjunction with the pTet promoter of an “inverter”; pTet will be “turned off” in the presence of arabinose.
PROCEDURE

Turn on heat bath to 42°C
Obtain 10 μL of nuclease free PCR water

Mix into cell 2 M of the titer plate
Set for 5 minutes

Put the mixture into the PCR tube

Nanodrop

Obtain 2 μL of DI water
Clean the lens with a kim wipe
Drop the 2 μL of DI water on the aperature of the nanodrop to blank

Repeat with a re-blank

Drop 2 μL of the mixture on the nanodrop
Measure on the nanodrop

Save as well 2M Plate 1 sp20
Hit measure

Bacterial Transformation of BBa_K542003

Purpose
Introduce a foreign plasmid into bacteria and to use those bacteria to amplify the plasmid in order to make large quantities of the plasmid. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.
The efficiency of the protocol is HIGHLY dependent on timing.
Conversions
100 ng of BBa_K542003 x 1 μL / 61.6 ng = 1.62 μL of gene
PROCEDURE

Start thawing competent cells (E. coli top 10) on ice

Competent cells are in the -80°C freezer

Add 100 μL of competent cells into a 1.5 mL centrifuge tube
Add 100 ng of DNA to the 1.5 mL centrifuge tube
Incubate on ice for 30 minutes
Heat shock cells in water bath for EXACTLY one minute

Make sure the water level is high energy for the tube to float
Adjust the temperature to 42°C

Incubate on ice for five minutes
Add 600 μL of psi broth to each centrifuge tube

Psi broth is in the -4°C freezer

Incubate centrifuge tubes in shaking incubator at 37°C at 200 rpm for two hours

Make three dilutions:

Obtain two fresh centrifuge tubes and label them with the appropriate dilution

1:10
1:100
1:1000

Add 90 μL of psi broth to each centrifuge tube BEFORE diluting
Add 10 μL of transformed cells to the 1:10 dilution

Mix the solutions by pipetting up and down multiple times

Take 10 μL from the 1:10 solution and place it in the 1:100 dilution

Repeat for the 1:1000 solution

Competent Cells Protocol

The goal of this protocol is to make E. coli competent meaning the cells can uptake plasmids. We do this chemically. The strain of E. coli should not contain any plasmids which is why the strain we used was TOP10. How we obtained TOP10 was through Doc. He has access to the freezer stocks of all the organisms in the microbiology department. For future reference when you ask for a specific strain you need an LB plate and a sterile inoculating stick. From there, Doc will do an EI (environmental isolate) streak. This plate needs to incubate at 37°C overnight. The next day you should do a quadrant streak in order to isolate colony forming units (CFU) and incubate the second culture overnight in the 37°C incubator to grow the CFU into a well-established culture for later use. We take CFU’s because it ensures us there is no contamination or other DNA present. It’s important to note that you want your colonies to be fresh.

In the future, the competent cells will be used to transform plasmids into E. coli. The reason for this is that the organisms we use are anaerobic. Therefore, E. coli (aerobe) is our work horse in which we use it to grow up our plasmids and makes the research easier without accounting for the difficulties that come with working with anaerobic cells.

Tip: Walk yourself through the protocol as if you were doing it. Have a teammate present and discuss each to step to ensure you have an understanding of how and why this protocol is done (we don’t do things without a purpose in the lab otherwise we are wasting our time). Prepare all the materials needed beforehand and collaborate with relevant persons to ensure lab tools will be accessible. If you are prepared then the results should be in your favor. Worst comes to show you will have precise details of your experiments and will easily be able to pinpoint mistakes that need to be fixed.

Materials:

Sterilized Items

1 test tube
1 side-arm flask

Media and Buffers

TFB1
TFB2
LB Liquid (>200 mL)
2 LB plates

Spectrometer
Bunsen Burner
Inoculating Loop
Shaking incubator availability

You need specific temperature and rpm’s

Green Bucket with ice

Should be ready when needed. Don’t interrupt your experiment to get ice

Pipette + Tips
Syringe (3 mL) + needle
Nitrile gloves
Box to store finish products in freezer

Culture E. coli (no plasmid)

Obtain from Doc as noted above
Stocks are in -80°C Freezer

Use syringe + needle to remove approximately 25 microliters and spread onto LB plate

Isolate a CFU
Fill a test tube with 10 mL of LB broth

Test tubes must be sterilized via autoclave

Inoculate test tube with a CFU

Do this step at about 5pm because when you check the OD (optical density) the next day, you don’t want the OD to be too high which can indicate the cells have entered a different phase in cell development.

Place inoculated test tube in shaking incubator (200 rpm; 37°C) overnight

Make sure it’s on the shaking platform, NOT the top stagnate shelf.

Take sterilized side-arm flask; fill it with 100 mL of LB broth and then inoculate it with 1 mL of culture (inoculated test tube in shaking incubator)

Be sure to pre-warm LB broth (place in incubator for 30 minutes or set out on bench) because the temperature shock may negatively impact the E. coli.
Use sterile techniques to transfer LB liquid to flask. Use syringe to top it off for accuracy

Place side-arm flask in shaking incubator (37°C; 200 rpm) for two hours
Label 30 centrifuge tubes

Include

Date
Your initials
Competent E. coli TOP 10 (assuming that is the strain)

Measure OD (440)

Absorbance should be at 0.5; keep incubating and checking until desired absorbance is obtained

DON’T let the absorbance exceed 0.55

To measure the OD, place the side-arm of the flask into the spectrometer slot

USE para film to keep the top covered and prevent spillage
Be sure the liquid is exposed to the side of the para film that is not exposed to the environment in order to prevent contamination

Cool culture on ice for 5 minutes
Transfer 1.5 mL of culture into 30 centrifuge tubes
Centrifuge tubes at 4000g (6100 rpm) for 5 minutes
Discard Supernatant

KEEP cells on ice the entire time

Add 450 microliters of TFB1 to re-suspend cells
Keep cells on ice for 1 hour
Centrifuge tubes at 4000g (6100 rpm) for 5 minutes
Discard Supernatant

KEEP cells on ice

Add 1000 microliters of TFB2 to re-suspend cells
Freeze cells immediately in -80°C freezer