|
|
| (16 intermediate revisions not shown) |
| Line 6: |
Line 6: |
| | <div class="gutter"> | | <div class="gutter"> |
| | <h1><span></html>Wet Lab Experiments<html></span></h1> | | <h1><span></html>Wet Lab Experiments<html></span></h1> |
| - | </html>{{{content}}}<html>
| |
| - | </div>
| |
| - | </section>
| |
| - | </div>
| |
| - |
| |
| | </html> | | </html> |
| | | | |
| - | Our 2 main wet lab projects were the transformation of Bacillus Subtilis and assembly of our plasmids using Gibson Assembly. | + | <p>Our two main wet lab projects were the transformation of Bacillus Subtilis and assembly of our plasmids using Gibson Assembly.</p> |
| | | | |
| - | We first conducted growth curve assays of our two strains (3A37 and 1A436) of Bacillus Subtilis.
| |
| | | | |
| - | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/9/94/GrowthCurve2.png" width="350" height="200" align="right"></div></html>
| |
| | | | |
| - | Electroporation Protocol
| + | '''Transformation of Bacillus Subtilis |
| | + | ''' |
| | + | <p>We first conducted growth curve assays of our two strains (3A37 and 1A436) of Bacillus Subtilis obtained from The Ohio State Bacillus Stock Center . </p> |
| | | | |
| - | Purpose
| + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/9/94/GrowthCurve2.png" width="550" height="350" align="center"></div></html> |
| - | Electroporation promises better transformation efficiency than starvation protocols and takes less time and is easier to execute. This protocol outlines how to use the Life Technologies Cell-Porator machine found in Bindley 222.
| + | |
| | | | |
| - | Solutions
| + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/e/e7/Electroporation_Picture.PNG" width="600" height="600" align="center"></div></html> |
| - | Growth medium: LB medium containing 0.5 M sorbitol
| + | |
| - | Washing solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol
| + | |
| - | Electroporation solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol
| + | |
| - | Outgrowth medium: LB medium containing 0.5 M sorbitol and 0.38 M mannitol
| + | |
| | | | |
| - | Materials
| + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/3/31/Electroporation_Picture_2.PNG" width="550" height="550" align="center"></div></html> |
| - | • Life Technologies Cell-Porator
| + | |
| - | • Cell-Porator chambers (4)
| + | |
| - | • Competent cells
| + | |
| - | • Micropipette and tips
| + | |
| - | • SOC solution
| + | |
| | | | |
| - | Methods
| |
| | | | |
| - | Making electrocompetent cells:
| + | '''Results''' |
| - | 1. Dilute an overnight culture of Bacillus subtilis 16-fold in growth medium and grow at 37 °C to an O.D.600 of 0.85-0.95.
| + | |
| - | 2. Cool the cells on ice-water for 10 min. and harvest by centrifugation at 4 °C and 5000 x g for 5 min.
| + | |
| - | 3. Wash cells four times in ice-cold electroporation medium.
| + | |
| - | 4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of
| + | |
| - | 1-1.3 x 1010 cfu/ml.
| + | |
| - | 5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.
| + | |
| | | | |
| - | Electroporation of cells:
| + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/9/91/SAM_0252.jpg" width="550" height="450" align="center"></div></html> |
| - | 1. Add 1 µl (50 ng/µl) plasmid DNA to 60 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Incubate for 1-1.5 min.
| + | |
| - | 2. Place ice water in chamber under cell holder rack
| + | |
| - | 3. Place electroporator chambers on ice
| + | |
| - | 4. Turn on voltage booster and controller using switches on back
| + | |
| - | 5. Set to these conditions:
| + | |
| - | 6. Voltage Booster: Level 4
| + | |
| - | 7. Charge rate: fast
| + | |
| - | 8. Volts: Low
| + | |
| - | 9. Capacitance: 330 | + | |
| - | 10. Pipette 20μL of competent cells with DNA between electrodes
| + | |
| - | 11. Fill all 4 slots in chamber
| + | |
| - | 12. Close chamber, plug in power source
| + | |
| - | 13. Make sure chamber selector is on the correct chamber
| + | |
| - | 14. Press up on the controller until voltage reaches 405
| + | |
| - | 15. At 405, switch from charge to arm and press trigger for 1 second
| + | |
| - | 16. Release trigger and switch arm to charge *If there is too much salt in the DNA, a pop will happen and the liquid will not be suspended between the electrodes
| + | |
| - | 17. Immediately add 1 ml outgrowth medium and incubate for 3 h at 37 °C.
| + | |
| - | 18. Plate onto selective LB agar plates and incubate overnight at 37 °C.
| + | |
| | | | |
| - | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/9/91/SAM_0252.jpg" width="350" height="200" align="right"></div></html>
| |
| | | | |
| | | | |
| - | Phytosiderrophore Production Assay
| |
| | | | |
| - | O-CAS Siderophore Assay
| |
| | | | |
| - | Introduction
| |
| - | The CAS assay includes inhibitory compounds that negatively impact the growth of gram-positive bacteria (e.g. Bacillus subtilis) and fungi. Therefore, the O-CAS assay must be used.
| |
| | | | |
| - | Materials
| + | Once we have functional circuits we will conduct the following Pytosidderophore Assay adapted from: |
| - | • 20% glycerol solution
| + | |
| - | • Chrome azurol S (CAS)
| + | |
| - | • HDTMA
| + | |
| - | • PIPES
| + | |
| - | • Agarose
| + | |
| | | | |
| - | Procedure
| + | Pérez-Miranda, S., Cabirol, N., George-Téllez, R., Zamudio-Rivera, L. S., & Fernández, F. J. (2007). O-CAS, a fast and universal method for siderophore detection. Journal of microbiological methods, 70(1), 127-131. |
| - | 1. Plates containing growth medium were stored at 28°C for 24h
| + | |
| - | 2. Bacteria were preserved in 20% glycerol solution at -20°C
| + | |
| - | 3. The CAS medium is prepared as follows:
| + | |
| - | a. Chrome azurol S (CAS) 60.5 mg
| + | |
| - | b. hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg
| + | |
| - | c. Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g
| + | |
| - | d. 1 mM FeCl3• 6H2O in 10 mM HCl 10 mL.
| + | |
| - | e. Agarose (0.9%, w/v) was used as gelling agent
| + | |
| - | 4. Apply 10mL of medium over standard Petri dish (or 30mL over large Petri dish)
| + | |
| - | 5. Color will change after 15 min if siderophores are detected
| + | |
| | | | |
| - | Paper
| |
| - | http://ac.els-cdn.com/S0167701207001315/1-s2.0-S0167701207001315-main.pdf?_tid=0f6ae27a-f585-11e3-a635-00000aacb361&acdnat=1402943939_6d4bbe673ecc5dfa11e7309507a2c4e8
| |
| | | | |
| | + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/8/86/Phytosiddrophore_Assay2.PNG" width="550" height="550" align="center"></div></html> |
| | | | |
| | | | |
| | + | '''Gibson Assembly |
| | + | ''' |
| | + | <p>We synthesized our two plasmids using Integrated DNA Technologies (IDT). We currently have them as gBlocks and are trouble shooting the Gibson Assembly.</p> |
| | | | |
| | | | |
| - | We synthesized our 2 plasmids using Integrated DNA Technologies (IDT). We currently have them as gBlocks and are trouble shooting the Gibson Assembly.
| + | <html> |
| | + | </div> |
| | + | </section> |
| | + | </div> |
| | + | |
| | + | </html> |
Wet Lab Experiments
Our two main wet lab projects were the transformation of Bacillus Subtilis and assembly of our plasmids using Gibson Assembly.
Transformation of Bacillus Subtilis
We first conducted growth curve assays of our two strains (3A37 and 1A436) of Bacillus Subtilis obtained from The Ohio State Bacillus Stock Center .
Results
Once we have functional circuits we will conduct the following Pytosidderophore Assay adapted from:
Pérez-Miranda, S., Cabirol, N., George-Téllez, R., Zamudio-Rivera, L. S., & Fernández, F. J. (2007). O-CAS, a fast and universal method for siderophore detection. Journal of microbiological methods, 70(1), 127-131.
Gibson Assembly
We synthesized our two plasmids using Integrated DNA Technologies (IDT). We currently have them as gBlocks and are trouble shooting the Gibson Assembly.
"
