Revision as of 19:53, 18 September 2015

The most important recipe for effective research and reproducible results are reliable protocols.

On this page we are providing several protocols that we have used over the course of our project.

Culturing

Cloning

B0034_Insertion-Mutagenesis[+]

This year our iGEM team tested and applied the RDP assembly method by Synbiota. We used it to assemble the polycistronic methanol conversion plasmid and to design a monocistronic diversity library to screen for a more efficient methanol uptake plasmid.

RDP parts

RDP parts can be assembled successively to build new devices or whole new circuits with unique functions. The sequence of every RDP part begins and ends with a 4 bp long 5' overhang.

There are four differnet kinds overhangs:

X: 5'- GATG -3'

X': 5'- CTAC -3'

Z: 5'- CGGC -3'

Z': 5'- GCCG -3'

As one can easily notice, X and X' as well as Z and Z' are homologous. A RDP part can have one of two possible orientations:

X - Z' or Z - X'

Usually, you can convert a certain sequence into a RDP part by PCR amplification. You have to use forward and reverse primers with extension containing BsaI restriction sites prior to the overhang sequence you want to introduce.

In the end, the 4 bp overhangs arise from a BsaI digest of the amplification product.

RDP parts with a length of <100 bp can be created by annealing synthesized oligos (HPLC purification recommended).

Assembling the parts

But how are RDP parts joined together to form whole new circuits? Synbiota's RDP assembly kit provides so called anchors. Anchors are linear DNA fragments, contain an antibiotics resistance gene and are bound to magnetic beads. At their downstream ends, anchors have either X' or Z' overhangs.

You can add any desired RDP part with the appropriate overhangs to the anchors that were diluted in an eppi. Matching 4 bp overhangs will be joined by T4 ligase in the reaction mix.

By applying a magnet to the eppi, you can collect all magnetic beads with the bound DNA and simply discard the used reaction mix. After washing and diluting the magnetic beads again, you can add your next RDP part. After assembling the RDP parts, you'll have to add a so called "cap". It is also provided in Synbiota's assembly kit and contributes an origin of replication.

RDP assembly
bla

You can add your first RDP part to the anchor. T4 ligase in the reaction mix will ligate the homologous overhangs
Before you can add the next RDP part, you have to collect the magnetic beads, discard the used reaction mix and then wash and resuspend the beads again.
In the end, the cap is added to terminate the assembly

Finally, anchor and cap can be joined to form a circular plasmid. By choosing a certain anchor and cap for the assembly you determine the resistance marker and copy number of the resulting plasmid.

Circularizing the assembly product
After releasing the finished RDP circuit from the magnetic bead, anchor and cap join to form a circular plasmid.

RDP plasmids can again be digested with BsaI and NotI to extract the assembled RDP parts as one composite RDP part.

Extracting a composite RDP part
To extract the assembled RDP parts as one composite RDP part, the plasmid is digested with BsaI to introduce the 4 bp overhangs and NotI to prevent the interference of the backbone fragment in future assembly steps.

Experiences

We experienced the RDP assembly method to be really easy to handle. Both the in silico work to design new RDP circuits and the efficiency of the method itself convinced us. Thus, RDP assembly can be a suitable alternative to other assembly methods like Gibson Assembly or CPEC.

Analytics

Acid Hydrolysis[+]

Cell Lysis[+]

Electrophoresis[+]

Glycogen Kit[+]

Glycogen Assay Protocol^[8]

1. Sample preparation

Homogenize 10⁶ (animal) cells with 200 µL dH₂O on ice.
- approximately 10⁹ E.coli cells (1 ml culture with OD 1.25) ^[9] ^[10]
Sonicate cells
Boil homegenates for 10 minutes.
Spin/centrifuge at 18 000 g for 10 minutes. The supernatant is now ready for assay!
Add 2-50 µL samples to a 96-well pate.
Adjust volume to 50 µL/well with Hydrolysis Buffer

Notes:

For samples having glucose background, prepare well(s) containing same amont of sample as in the test well as background control.
(Accurate determination of Glycogen in the test samples is best determined via spiking samples with a known amount of standard (example given 0.8 µg))

2. Standard Curve preparation

Dilute the glycogen standard to 0.02 mg/mL by adding 10 µL of the Standard to 990 µL of distilled water, mix well.
Add 0, 2, 4, 8, 10 µL to a series of wells.
Adjust volume to 50 µL/well with Hydrolis Buffer to generate 0, 0.04, 0.08, 0.12, 0.16, and 0.2 µg per well

3. Add (1 µL) Hydrolysis Enzyme mix to Standards and samples and mix well.

for dilution samples, dilute with hydrolysis buffer

Incubate for 30 minutes at room temperature.

Note:

If glucose is present in your sample, you may do a glucose control without addition of hydrolysis enzyme to determine the level of glucose background.

4. Reaction Mix

Mix enough reagents for the number of samples and standards to be performed
For each well prepare a total 50 µL Reaction Mix.
- Dev. Buffer: 48.7 µL
- Dev. Enzyme Mix: 1 µL
- OxiRed Probe: 0.3 µL
Add 50 µL of the Reaction Mix to each well containing the Glycogen Standard or samples, mix well.

5. Measurement

Incubate the reaction for 30 minutes at room temperature protected from light.
- Fluorescence (Ex/Em = 535/587 nm)

6. Calculation

Correct background by subtracting the 0 Glycogen Standard sample value (see 2.) from all readings.
Apply sample readings to the standard curve to get B µg of glycogen.

Sample c_(Glycogen) = B / V * D µg/µL

B is the amount of Glycogen from Standard curve (µg)

V is the sample volume added into the reaction well (µL)

D is the sample dilution factor

For spiked samples, correct for any sample interference by subtracting the sample reading from spiked sample.

For Spiked samples:

B = [OD_(sample corrected) / OD_(sample+glycogen Standard (corrected)) - OD_(sample corrected)] * Glycogen Spike (µg)

Notes about Glycogen:

Glycogen molecular size: 60 000 molecules
- Molecular Weight (MW) ~ 10⁶-10⁷ daltons
- Glucose MW: 180.16 g/mol

Glycogen Assay Protocol short version

1. Sample preparation

Homogenize 10⁶ cells with 200 µL dH₂O on ice.
Boil homegenates for 10 minutes.
Spin/centrifuge at 18 000 g for 10 minutes. The supernatant is now ready for assay!
Add 2-50 µL samples to a 96-well pate.
Adjust volume to 50 µL/well with Hydrolysis Buffer

2. Standard Curve preparation

Dilute the glycogen standard to 0.02 mg/mL by adding 10 µL of the Standard to 990 µL of distilled water, mix well.
Add 0, 2, 4, 8, 10 µL to a series of wells.
Adjust volume to 50 µL/well with Hydrolis Buffer to generate 0, 0.04, 0.08, 0.12, 0.16, and 0.2 µg per well

3. Add (1 µL?) Hydrolysis Enzyme mix to Standards and samples and mix well.

Incubate for 30 minutes at room temperature.

4. Reaction Mix

Mix enough reagents for the number of samples and standards to be performed
For each well prepare a total 50 µL Reaction Mix.
- Dev. Buffer: 48.7 µL
- Dev. Enzyme Mix: 1 µL
- OxiRed Probe: 0.3 µL
Add 50 µL of the Reaction Mix to each well containing the Glycogen Standard or samples, mix well.

5. Measurement

Incubate the reaction for 30 minutes at room temperature protected from light.
- Fluorescence (Ex/Em = 535/587 nm)

Pre-extraction of glycogen

Another way to subtract the glucose background, is the pre-extraction of glycogen
This procedure removes the majority of glucose background with minimal effect on glycogen

Take tissue or cells to a final content of 30-50 % in 30 % KOH
Heat to 100 °C for 2 h
Cool and add 2 volumes of 95% ethanol to precipitate crude glycogen
Centrifuge (16 000g for 10 min) and collect the precipitate
Suspend the pellet in a minimal amount of distilled water (25 ml original cell culture -> 5 ml were enough)
Acidify sample to pH3 with 5 M HCl
Add 1 volume of ethanol to reprecipitate
Repeat steps 4-7 two more times, then let sample dry over night (pre-weigh container for drying)
The dried material can be weighed and dissolved for analysis

References

↑ Bryksin AV, Bachman HN, Cooper SW, Balavijayan T, Blackstone RM, Du H, Jenkins JP, Haynes CL, Siemer JL, Fiore VF, Barker TH. One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick. ACS Synth Biol. 2014 Dec 19;3(12):956-9. doi:10.1021/sb500047r. PubMed PMID: 25524097; PubMed Central PMCID: PMC4277749.
↑ Quan, Jiayuan, and Jingdong Tian. “Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways.” Ed. Paulo Lee Ho. PLoS ONE 4.7 (2009): e6441. PMC. Web. 16 Sept. 2015.
↑ https://j5.jbei.org/j5manual/pages/22.html
↑ https://www.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly
↑ http://www.geneious.com
↑ http://parts.igem.org/Help:Transformation_Protocol Protocol of the iGEM HQ
↑ Blank Lars, Protocol for 13C Tracer Experiments
↑ [http://www.biovision.com/manuals/K646-100.pdf Glycogen Assay Kit Manual]
↑ https://www.thermofisher.com/de/de/home/references/ambion-tech-support/rna-tools-and-calculators/macromolecular-components-of-e.html
↑ http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp
↑ Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochemical Journal, 55(3), 416-421.
↑ Kleeberg & Klinger. 1982. Sensitive formaldehyde determination with Nash's reagent and a tryptophan reaction. Journal of Pharmacological Methods, 8(1), 19-31.
↑ S. K. Meur, V. Sitakara Rao, and K. B. De. Spectrophotometric Estimation of Reducing Sugars by Variation of pH. Z. Anal. Chem. 283, 195-197 (1977)

▲

[1] Bryksin AV, Bachman HN, Cooper SW, Balavijayan T, Blackstone RM, Du H, Jenkins JP, Haynes CL, Siemer JL, Fiore VF, Barker TH. One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick. ACS Synth Biol. 2014 Dec 19;3(12):956-9. doi:10.1021/sb500047r. PubMed PMID: 25524097; PubMed Central PMCID: PMC4277749.

[2] Quan, Jiayuan, and Jingdong Tian. “Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways.” Ed. Paulo Lee Ho. PLoS ONE 4.7 (2009): e6441. PMC. Web. 16 Sept. 2015.

[3] ttps://j5.jbei.org/j5manual/pages/22.html

[4] ttps://www.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly

[5] ttp://www.geneious.com

[6] ttp://parts.igem.org/Help:Transformation_Protocol Protocol of the iGEM HQ

[7] Blank Lars, Protocol for 13C Tracer Experiments

[8] [http://www.biovision.com/manuals/K646-100.pdf Glycogen Assay Kit Manual]

[9] ttps://www.thermofisher.com/de/de/home/references/ambion-tech-support/rna-tools-and-calculators/macromolecular-components-of-e.html

[10] ttp://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp

[11] Nash. 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochemical Journal, 55(3), 416-421.

[12] Kleeberg & Klinger. 1982. Sensitive formaldehyde determination with Nash's reagent and a tryptophan reaction. Journal of Pharmacological Methods, 8(1), 19-31.

[13] S. K. Meur, V. Sitakara Rao, and K. B. De. Spectrophotometric Estimation of Reducing Sugars by Variation of pH. Z. Anal. Chem. 283, 195-197 (1977)

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@@ Line 238: / Line 238: @@
 *Set up the reaction according to the table below on ice (2-3 fragment assembly).
-*Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
+*Incubate samples in a thermocycler at 50 °C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20 °C for subsequent transformation.
 *Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.

Ingredient	Concetration
KH2PO4	227 mM
Na2HPO4	472 mM
NaCl	85 mM
NH4Cl (standard/low)	187 mM / 22.4 mM

Component	Final Concetration	Concetration in stock solution
Iron(III) chloride	50 mM	13,515 mg/L
Calcium chloride	20 mM	2,220 mg/L
Manganese(II) chloride	10 mM	1,258 mg/L
Zinc sulfate	10 mM	1,615 mg/L
Cobalt(II) chloride	2 mM	260 mg/L
Copper (II) chloride	2 mM	269 mg/L
Nickel(II) chloride	2 mM	259 mg/L
Sodium molybdate	2 mM	412 mg/L
Sodium selenite	2 mM	346 mg/L
Boric acid	2 mM	124 mg/L
Hydrochloric acid	1 mM	20 ml

Total Amount of Fragments	0.02-0.5 pmols
Gibson Assembly Master Mix (2X)	10 µl
Deionized H2O	10-X µl
Total Volume	20 µl

task	time	temperature
incubate	30'00	4
shock	1'00	42
incubate	5'00	4

Difference between revisions of "Team:Aachen/Notebook/Protocols"

Revision as of 19:53, 18 September 2015

Culturing

Lysogeny-Broth (LB Medium)[+]

M9 Medium[+]

Overnight Cultures[+]

SOC Medium[+]

Cloning

B0034_Insertion-Mutagenesis[+]

CPEC[+]

Gibson Assembly[+]

Genomic Amplification[+]

Transformation[+]

Plasmid Preparation[+]

RDP Assembly[+]

Analytics

Acid Hydrolysis[+]

Cell Lysis[+]

Electrophoresis[+]

Glycogen Kit[+]

Iodine Staining[+]

Nash Assay[+]

Priciple of detection

Components ^[12]

Procedure

SDS-PAGE[+]

Dinitrosalicylic Acid Staining[+]

References

Ingredient	Stacking Gel (1mL)	Running Gel (5 mL; 15%)
H₂O	0,72 mL	1,73 mL
1,5 M Tris	0,13 mL (pH 6,8)	1,3 mL (pH 8,8)
10% Ammoniumperoxodisulfat	0,01 mL	0,05 mL
10% N,N′-Methylenbisacrylamid	0,13 mL	0,05 mL
30% Acrylamid	0,13 mL	1,87 mL
TEMED	0,001 mL	0,002 mL

Ingredient	Concentration
I2	5 mM
KI	5 mM

Difference between revisions of "Team:Aachen/Notebook/Protocols"

Revision as of 19:53, 18 September 2015

Culturing

Lysogeny-Broth (LB Medium)[+]

M9 Medium[+]

Overnight Cultures[+]

SOC Medium[+]

Cloning

B0034_Insertion-Mutagenesis[+]

CPEC[+]

Gibson Assembly[+]

Genomic Amplification[+]

Transformation[+]

Plasmid Preparation[+]

RDP Assembly[+]

Analytics

Acid Hydrolysis[+]

Cell Lysis[+]

Electrophoresis[+]

Glycogen Kit[+]

Iodine Staining[+]

Nash Assay[+]

Priciple of detection

Components [12]

Procedure

SDS-PAGE[+]

Dinitrosalicylic Acid Staining[+]

References

Components ^[12]