Team:Uniandes Colombia/WetLab

iGEM Uniandes-Colombia

Team Project Wet Lab Dry Lab Hardware Human Practices Collaborations -

Wet Lab

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Safety
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Protocols
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Notebook
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Biosafety Provisions

Institutional Regulations

Our project safety procedures are governed by the general regulations of the University regarding the proper standard rules and safety guidelines for laboratory practices. Teaching and research laboratories in the university are specially regulated accordingly to the dependence they belong to. In our case, the Faculty of Sciences Department of Biological Sciences and Department of Physics. The specific guidelines for the laboratory work at the Department of Biological Sciences are available online (in Spanish only) Biosafety Guidelines. Our main workspaces are the LAMFU and the Biophysics facilities, BSL2 research laboratories, but also we occasionally work on several BSL1 teaching laboratories (Department of Biological Sciences).



Institutional Biosafety Committees

There is the Parietal Committee for Occupational Health (COPASO), regulated by the Colombian Constitution (Resolution Number 2013, Year 1989). The information regarding the functions and regulations of the COPASO committee is available online (Spanish only). There is also the Bioethical Committee of the Faculty of Sciences, to whom we have presented our project proposal. Although we are still waiting for a response from the committee, our research methods and safety precautions are well within the regulations of the Committee.



Biosafety Training & Work Precautions

All team members were required to attend safety training focused on: • General biosafety principles. • The proper use of containment equipment (Biological safety (laminar flow) cabinets and delimited working space when required) as well as personal protective equipment (PPE) (White coat and gloves). • Risk assessment of experiments involving: Recombinant DNA, ethidium bromide, other biohazardous materials. • Proper Biological waste disposal. Our main concern was to work with the appropriate safety guidelines for working in both research and teaching laboratories (BL1-BL2) in order to minimize exposure to hazardous agents. We also work with the participating laboratories´ procedures to minimize waste and encourage use of reusable glassware instead of disposable plastic.



Safety Considerations:

Hazardous reagents

and Biohazards


Health Risks & Personal Safety

Working in the lab always has implicit risks, but we have taken action to minimize them. Some of the precautions we take are listed here: • The microorganisms are grown in large petri dishes that are sealed completely during bacterial growth, thus preventing their escape. • Equipment such as biological safety cabinet class I and laminar flow cabinets are used regularly to minimize the possibility of spread of microorganisms by recently trained students. • All students involved with the project were informed of the established security arrangement and signed a commitment to strictly comply with the rules. • Also, prior to the beginning of the experimental procedures, all of the team members were given a course on biosafety, focused mainly on biological hazards, chemical reagents and general BSL-1 laboratory safety concerns and security procedures. The system design tests are conducted within a laboratory of biosafety level 1, and we use the following materials and microorganisms strains:



Known toxic chemical reagents and hazardous physical agents:

Ethidium bromide

Since ethidium bromide is a very toxic agent, we have changed our nucleic acid staining molecules to SYBR® Safe. This claims to be significantly less toxic and better for the environment. Indeed, according to the manufacturer, it may be disposed directly into wastewater drainage systems (not a risk we cared to take, though).


Ultraviolet light

It is a type of electromagnetic radiation with a shorter wavelength shorter than that of visible light. We use UV in the biological safety and laminar flow cabinets for sterilization and decontamination purposes. Also, UV radiation is used for visualization of the stained DNA in the electrophoretic gels. In order to prevent accidental exposition to UV radiation, precautions are taken and when necessary, special safety lenses are used. We always use UV-blocking shields when visualizing electrophoretic gels in UV light. All other toxic and chemicals will be handled to avoid direct contact, and observing the proper safety procedures, additionally, all chemicals reagents and biological materials will be disposed only in the designated biohazard receptacles, following the regulations for Guidelines for Integral Solid Waste Management of the Medical and Occupational Health Department at the Universidad de los Andes.





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Protocols


Agarose gel

  1. Weigh 0,3 g of agarose.
  2. Add 30 mL of TAE 1X.
  3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
  4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
  5. When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
  6. Pour the solution into the bed and clear all its bubbles with a tip. Put a piece of paper on top of it and let it polymerize.
  7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.
Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.


LB medium (1L liquid)

  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water



LB medium (solid, 1L = 50 dishes)

  • 15 g agar agar
  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water

For selective medium, suplement with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin ).


Electrocompetent cells

  1. Divide the ON culture in 50 mL falcon tubes (we always used ''E. coli'' TOP10).
  2. Centrifuge 8000 rpm x 10 min.
  3. Discard supernatant.
  4. Resuspend everything with water in two falcons.
  5. Centrifuge again.
  6. Discard supernatant and resuspend with water. Wash with water two more times.
  7. Centrifuge again.
  8. Discard supernatant.
  9. Resuspend with glycerol with water. Glycerol 10 %.
  10. Centrifugue.
  11. Repeat steps 8-10.
  12. Discard supernatant and divide what is left in eppendorfs.
CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).


Genome extraction

For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to manufacturer's instructions.


Plasmids extraction

For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to manufacturer's instructions.


Transformation by electroporation

  1. Mix 40 µL of electrocompetent cells with 4 µL of DNA (we used iGEM BioBricks resuspended in 20 µL of miliQ water ).
  2. Incube the cuvettes for electroporation on ice and the above mix as well.
  3. Electroporate into the cuvette.
  4. Add (as quick as possible) 200 µL of LB medium.
  5. Incubate for 30 min at 37 °C .
  6. Plate bacteria over sumplemented LB medium.
  7. Incubate at 37 °C, 24 h.
  8. Use isolated colonies to check the correct insertion.



Biobrick Assembly

The digestion and ligation of the BioBricks were carried out using the Biobrick Assembly Kit, New England Biolabs Inc. The protocols for both procedures can be found here.

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Lab Notebook

 

Wednesday 05/20 by Sofia, Valentina, Santiago and Eitan

Advances:

  • The enzymes stock was initiated
  • Chemocompetent cells were made.
  • Chemocompetent cells were all used.
  • Buffer TSS was made.
  • The following parts were resuspended:
    • LuxI
    • LacI
    • pLux
    • pLac
    • aaiA
    • LuxR (part from 2015)
    • LuxR (part from 2014)
  • All the parts mentioned above were transformed and streaked in plates with Clor antibiotic.
  • We shared a chocolate bar because we did an excellent job.

Things left to do:

  • Pass every successful transformation to liquid LB medium.
  • Make liquid LB medium.
  • Finish the enzyme stock.
  • Pick all the equipment from sterilization.

 

Thursday 05/21 by Eitan.

Results from previous work session:

  • YAYYY nothing worked :D
  • Chloramphenicol was defective so the plates were contaminated.

Advances:

  • 15 new plates with chloramphenicol and another 15 with ampicillin were requested
  • 500ml of LB liquid medium was requested.

Things left to do:

  • Make a pass of E.coli (alfa) for ON
  • Make chemocompetent cells from↑

 

May 22. Pablo commandeering the lab

Results from previous work session: -

Advances:

  • Dishes were picked up at sterilization dept, left in Q502 fridge. LB with chloramphenicol and ampicillin.
  • LB liquid medium WAS NOT picked up from ster. dept.
  • 100 µL of transformed cells (see 05/20) were plated onto chloramphenicol LB. 7 plates used. Incubating at 37ºC.
  • Buffer needed for chemocompetent cell preparation still solid. ON not prepared.
  • Prepared TSS buffer again.

Things left to do:

  • Pick up LB liquid medium at sterilization dept.
  • Check 7 plates with transformed bacteria for colonies. Miniprep them.
  • Make a pass of E.coli (alfa) for ON once needed buffer is liquid.
  • Make chemocompetent cells from the ON.

May 23 Sofía and Pablo

Results from previous work session:

  • 7 plates had no colonies. Bacteria probably did not transform.

Advances: —

Things left to do:

  • Pick up LB liquid medium at sterilization dept.
  • Make a pass of E.coli (alfa) for ON once needed buffer is liquid.
  • Make chemocompetent cells from the ON.
  • Try to transform bacteria again.

 

May 25 Pablo rockin' it

Results from previous work session:

  • TSS buffer is liquid

Advances:

  • Suspended 1 E.coli DH5-alpha colony from plate in fridge in 10 mL of LB. Incubating ON inside shaker.
  • Picked up LB liquid medium at sterilization dept.

Things left to do:

  • Make chemocompetent cells from the ON.
  • Try to transform bacteria again.
  • Pray for a miracle.

May 26 Santiago, Sofia and Eitan.

Results from previous work session:

  • ON grew just fine

Advances:

  • 52 Chemocompetent cells were made.
  • TSS buffer was stored in the stock (4°)
  • Liquid culture of E.cherry for transformation control was made.
  • DH5A E.choli stock was replaced.

Things left to do:

  • Miniprep E. cherry (it has Amp resistance!!!)
  • Select a constitutive promoter from the registry (with high basal expression).
  • Transform the 7 parts suspended in may 20th plus the plasmid from E.cherry as control.
  • Run a gel with different parts for teaching reasons

 

May 27 Andres (“Santiago”), Pedro (“kevin”) and Eitan-Senseí.

Results from previous work session:

  • E.Cherry grew just fine

Advances:

  • E.Cherry was miniprepped
  • Constitutive promoter BBa_J23119 (PromC from now on) was resuspended
  • Tae buffer was sended to sterilization dept.
  • The Following parts were Transformed:
    • LuxI.
    • LacI.
    • pLux.
    • pLac.
    • aaiA.
    • LuxR (part from 2015).
    • LuxR (part from 2014).
    • PromC.
    • Transformation positive control (E.Cherry).

Things left to do (May 28):

  • Transform linearized plasmids.
  • Pick up the Tae buffer.
  • Pass to liquid medium the transformed cells.
  • Make more Petri dishes (15 for each antibiotic).

 

May 28 Pedro and Santiago

Results from previous work session:

  • 8 plates had no colonies.
  • Plate with E. Cherry (our control) did grew. Proves competent cells and transformation protocols do work.

Advances:

  • We reviewed our methodology.
  • Turns out we were making a “Bacterial Holocaust” with the insane amount antibiotics we were using.
  • The old plates were returned to sterilization.
  • New plates were prepared with the appropriate concentration of antibiotics.

Things left to do:

  • Pick the new plates from the sterilization department.
  • Make a new assay to determine if the problem were in fact the antibiotics doing the following experiment:
    • Choose a random part from the iGEM distribution kit. (check that it has no use for us and that it uses the same plasmid as our resuspended parts.
    • Resuspend it in 10uL.
    • Transform 4 E.coli using plasmid volumes of 1, 2, 3 and 4 uL.
    • Plate them.
    • Upload results.

June 01 Santiago and Eitan.

Results from previous work session:

  • Concentration assay didn’t work.

Advances:

  • the following parts were transformed:
    • 1A plate 1 form 2014
    • 1B plate 1 from 2015
  • The following parts were plated:
    • Concentration assay with 1, 3 and 4 uL again in case they expressed their genes in the weekend (hopefully)
    • LuxR from 2014 in the right medium
    • Part 1A from 2014 as control for the antibiotic (chloramphenicol).
    • Part 1B from 2015 was plated in the way of a antibiogram.

Things left to do:

  • Report results.
  • Pick up the mediums with different concentration of antibiotic:
    • 25 ug/mL
    • 20 ug/mL
    • 15 ug/mL
    • 10 ug/mL
    • 5 ug/mL
  • Plate part 1B from 2015 in these new mediums to identify the right quantity of chloramphenicol needed.

 

June 04 Santiago and Gabriela.

Results from previous work session:

  • Antibiogram showed an inhibition halo with no gradient. This means there were no transformed colonies and the antibiotic is working well.
  • None of the transformations were successful.

Advances:

  • Sterile pipette points picked up
  • 5 plates with different antibiotic concentrations picked up.
  • Part 1B-2015 plated on these 5 plates.
  • iGEM Transformation Efficiency Kit parts plated.

Things left to do:

  • Report results of 5 plates.
  • Report Transformation Efficiency Kit results.

 

June 04 Santiago and Pedro.

Results from previous work session:

  • No successful transformations either from 5 plates or from Transformation Efficiency Kit.

Advances:

  • Took pipette tips to sterilization
  • Ordered 30 agar plates with LB and chloramphenicol
  • Transformed all 7 parts into chemocompetent cells developed with new CaCl2 protocol
  • Transformed 5 of 7 parts into chemocompetent cells developed with TSS protocol

Things left to do:

  • Pick up plates and pipette tips from sterilization dept.
  • Check results of CaCl2 transformations
  • Transform luxR from the ‘14 distribution and aaiA into TSS chemocompetent cells.

 

June 09

Results from previous work session:

  • Pipette tips and 30 plates picked up at sterilization dept. (under my name)

Advances:

  • Solid LB medium components were and taken to sterilization, 20 plates
  • Liquid LB medium was weighed and taken to sterilization. 500 mL
  • Parts 1B and 1C from plate 1 f the ‘15 distribution were transformed into both TSS and CaCl2 chemocompetent cells. Transformed cells were plated onto LB+chloramphenicol.
  • 1C transformed cells (both TSS and CaCl2) were also plated onto LB plates with no antibiotic as a control
  • ON of DH5 alpha cells prepared for electrocompetent preparation

Things left to do:

  • Report results of transformations and control
  • Measure OD at 600 nm. If at 0.6, continue electrocompetent cell preparation protocol
  • Pick up plates and liquid LB from sterilization dept.

 

June 11 Pedro, Andrés, Samuel

Results from previous work session:

  • Transformations did not grow. Control grew.
  • OD reached 0.6 at 1:00 pm

Advances:

  • Picked up plates and liquid LB from sterilization dept.
  • Electrocompetent cells were prepared (26 Eppendorf tubes of 50 µL)
  • 400 mL of liquid LB medium were weighed
  • Eppendorfs were taken to sterilization
  • 3 attempts of electroporation transformation were carried out and plated on LB+chloramphenicol (20 µL were plated). Part 1D of plate 1 (‘15) was used. 3 µL of DNA were used (4 for the tube marked “a”).
  • A fresh culture of DH5 alpha was prepared and stored in fridge.

Things left to do:

 

June 12 Diego, Gabriela and Pablo

Results from previous work session:

  • One of the transformations showed 2 colonies!

Advances:

  • Part 1B of plate 5 (‘15) (a YFP2 reporter) was extracted and transformed using iGEM’s chemocompetent transformation protocol (http://parts.igem.org/Help:Protocols/Transformation), using both TSS and CaCl2 chemocompetent cells. The remaining extracted DNA was stored in the -30ºC freezer in the box marked “Silvia Cañas/Plásmidos nuevos”. The cells were plated onto LB+chloramphenicol agar.
  • YFP2 and one of the Transformation Efficiency RFP parts (20 pg/mL) were transformed into electrocompetent cells. The cells were plated onto LB+chloramphenicol agar.
  • Controls of both the chemo and electrocompetent transformations were plated onto LB agar without antibiotic.
  • A culture of one of the colonies from the plate transformed on June 11 was prepared on liquid LB.

Things left to do:

  • Report transformation results.
  • Report control results
  • Miniprep colony culture?

 

June 13 Sofía and Pablo

Results from previous work session:

  • One of the electrocompetent transformation plates grew 3 colonies, but contamination is suspected.
  • Both electrocompetent transformation controls grew.
  • CaCl2 chemocompetent control did not grow. TSS chemocompetent control showed suspicious growth only at edge of plate... contamination suspected.

Advances:

  • All plates stored in fridge for the weekend

Things left to do:

  • Miniprep colony culture?
  • Repeat CaCl2 control, confirm whether or not CaCl2 cell stock is dead
  • Repeat TSS control, confirm whether or not TSS cell stock is dead
  • Culture one of the colonies from the plate transformed on June 12 on liquid LB
  • Measure distribution DNA concentration using NanoDrop

 

June 16 Gabriela and Santiago

Results from previous work session: –

Advances:

  • Part 1B (plate 1, 2015)’s concentration was determined to be 95.7 ng/µL by NanoDrop.
  • 2 tubes of LB were inoculated with pink colonies from June 12’s electrocompetent transformants. One tube contained 25 µg/mL chloramphenicol.
  • LB liquid media was prepared
  • 2 attempts at electroporation transformation were carried out with 50 pg/µL RFP from the Transformation Efficiency Kit.
  • 500 mL SOB medium were prepared according to http://parts.igem.org/SOB
  • A 20% glucose solution was prepared in order to produce SOC medium as per http://parts.igem.org/SOC

Things left to do:

  • Sterilize glucose solution
  • Pick up media from sterilization dept.
  • Check for transformants in today’s transformations
  • Prepare chemocompetent cells with SOC medium. Try less centrifugation as Juan Manuel Pedraza suggested.
  • Buy a new Sharpie for the lab :(

 

June 17 Pedro & Andrés

Results from previous work session:

  • No transformants in yesterday’s work. Yet again.
  • The tubes inoculated with transformants showed no pigment production.

Advances:

  • Picked up media from sterilization dept.
  • Sterilized glucose solution
  • DH5 alpha cells were inoculated into LB as an overnight culture to prepare chemocompetent cells tomorrow.

Things left to do:

  • Prepare chemocompetent cells with SOC medium. Try less centrifugation as Juan Manuel Pedraza suggested.

 

June 18 Pedro, Andrés, Samuel & Pablo

Results from previous work session:

  • Cultures in tubes still show no pigment.

Advances:

  • Centrifuged tubes to check for pigment in pellet. None found. Tubes discarded.
  • A contamination was discovered in two pure LB control plates used in transformations. Staphylococcus aureus is suspected. Contaminant was streaked onto plate out of Pablo’s curiosity.
  • A fresh plate of DH5 alpha was prepared in a new LB plate.
  • Chemocompetent cells were prepared. 27 Eppendorf tubes were centrifuged for 5 minutes as part of the protocol, 21 were centrifuged for 10 instead.
  • 2 chemocompetent transformations were carried out with the above cells. The cells prepared with 5 minute centrifugation were transformed with 2 µL of 10 pg/µL DNA from the Transformation Efficiency Kit, while the 10 minute centrifugation cells were transformed with 2 µL of 20 pg/mL DNA.
  • One electrocompetent transformation was carried out with 4 µL of 50 pg/µL DNA from the TE Kit.
  • All transformations were plated onto LB medium with chloramphenicol as well as pure LB as control, and stored at -30ºC.

Things left to do:

  • Pick up piptte tips from sterilization dept.
  • Check transformations, determine whether 5 or 10 minute centrifugation had greater efficiency.
  • Sterilize Eppendorf tubes.

 

June 19 Pedro & Samuel

Results from previous work session:

  • No transformants in any of yesterday’s attempts. Controls grow well as usual.
  • DH5 alpha pass grew but was found to be contaminated
  • Contamination, on the other hand, grew beautifully. Confirmed as almost certainly S. aureus: yellow pigment; small, point-like colonies. Probably from previous lab work by another researcher in Biophysics lab who worked with this bacteria.

Advances:

  • New DH5 alpha plate prepared.

Things left to do:

  • Store DH5 alpha plate in fridge (if not contaminated)

 

June 20 Pedro

Results from previous work session:

  • DH5 alpha plate grew well.

Advances:

  • DH5 alpha plate stored in fridge.

 

June 23 Pedro

Results from previous work session: –

Advances:

  • Plated RFP and YFP-transformed cells from June 12 onto LB+Chlor. and LB again.
  • Took pippette tips, Erlenmeyer flask, and Eppendorf tubes to sterilization
  • DH5 alpha ON culture inoculated for use tomorrow

Things left to do:

  • Pick up stuff from sterilization dept. tomorrow
  • Prepare chemocompetent cells



June 24

Results from previous work session:

  • Chemocompetent cells could not be prepared today.

Advances:

  • New DH5 alpha was inoculated as an ON culture for tomorrow.

Things left to do:

  • Prepare chemocompetent cells

 

June 25

Results from previous work session:

  • Chemocompetent cells could not be prepared today either.

Advances:

  • New DH5 alpha was inoculated as an ON culture for tomorrow.
  • 400 mL of 10% glycerol were prepared
  • a 300 mL flask was sterilized for use tomorrow

Things left to do:

  • Prepare chemocompetent cells

 

June 26

Results from previous work session: –

Advances:

  • 300 mL LB were inoculated with 300 µL of ON culture
  • Cells did not grow well. Will have to start again tomorrow
  • Electrocompetent cells were prepared using Cesar Medina’s suggestions.

Things left to do:

  • Prepare chemocompetent cells

 

June 30

Results from previous work session:

  • Chemocompetent cells could not be prepared today.

Advances:

  • Electrocompetent transformation was carried out with the help of the wonderful people at the LAMFU lab. One of their own pUC19 plasmids was used at 10 pg/µL concentration.

Things left to do:

  • Try new electrocompetent transformations.
  • Try NEB chemocompetent cells, which arrived today

 

July 1

Results from previous work session:

  • Electrocompetent transformations failed. Control grew well.
  • SOC medium suspected to be contaminated.

Advances:

  • Transformed NEB chemocompetent cells with part 1E, plate 1, 2015 according to NEB’s protocol.
  • Transformations were plated at 10 different dilutions in LB, from 10^-1 to 10^-10.
  • SOC was placed in shaker to check if contaminated

Things left to do:

  • Check transformations
  • Check SOC for growth

 

July 2 Santiago & Gabriela, Andrés and Pedro

Results from previous work session:

  • Electrocompetent transformations failed. Control grew well.
  • SOC medium suspected to be contaminated.

Advances:

  • Transformed 2 NEB chemocompetent cells with part 1F, plate 1, 2015 (2 µL) according to NEB’s protocol. Salmon sperm DNA was used in one of the two.
  • Transformed the same part into electrocompetent cells with salmon sperm DNA.
  • Plated all transformations, with their controls.
  • Plated NEB competent cells onto pure LB for safekeeping.
  • 2 tubes of SOC (10, 20 mL) were inoculated with DH5 alpha cells to culture ON

Things left to do:

  • Check transformations
  • Store NEB cell plate in fridge
  • Prepare chemocompetent cells with NEB strain
  • Plate a fresh pass of DH5 alpha cells

 

July 3 Samuel, Andrés, Pedro & Santiago

Results from previous work session:

  • All controls grew well. One transformation showed 6 colonies: the NEB chemocompetent cells without salmon sperm DNA.
  • SOC medium suspected to be contaminated.

Advances:

  • The cells that apparently were transformed were plated again onto LB+chloramphenicol to check transformation success
  • NEB chemocompetent cells were transformed with part 1B (plate 1, 2015) to check whether results are reproducible
  • 15 plates of pure LB were ordered, Eppendorfs were taken to sterilization
  • ON culture of DH5 alpha cells was prepared

Things left to do:

  • Prepare chemocompetent cells with NEB strain

 

July 6

Results from previous work session:

Advances:

  • Parts 2C and 2D (plate 1, 2015) were transformed using electrocompetent cells.
  • 2 500 mL Erlenmeyer flasks were sterilized in anticipation of tomorrow’s chemocompetent cell production.
  • A fresh pass of DH5 alpha cells was plated.
  • An ON culture was prepared for tomorrow’s chemocompetent cell production.

Things left to do:

  • Make chemocompetent cells

 

July 7 Pedro & Andrés

Results from previous work session:

  • There was no NEB strain ON culture to work with, so chemocompetent cells will be prepared tomorrow.

Advances:

  • An ON culture of NEB cells was prepared.
  • Bacteria were transformed with part 2E via electroporation.

Things left to do:

  • Make chemocompetent cells

 

July 8 Santiago

Results from previous work session:

  • –

Advances:

  • pUC19 and part 3A (p. 1, 2015) were transformed using NEB chemocompetent cells.
  • An environmental control plate was left in the incubator.

Things left to do:

  • Make chemocompetent cells
  • Check control plate

 

July 9

Results from previous work session:

  • –

Advances:

  • ON LB cultures of transformed pUC19 and part 3A cells inoculated. pUC19 media required ampicillin, part 3A chloramphenicol.

Things left to do:

  • Make a quicky ON from both ON cuktures
  • Miniprep
  • NanoDrop
  • Electrophoresis

 

July 10 Andrés and Pedro

Results from previous work session:

  • –

Advances:

  • NEB strain cells were inoculated into LB medium to prepare chemocompetent cells. However, growth was poor and procedure was aborted.
  • Quicky ON cultures of pUC19 and part 3A transformants prepared. Ampicillin concentration: 50 µg/mL. Chloramphenicol: 25 µg/mL.

Things left to do:

  • Miniprep both quicky ON cultures
  • Nanodrop the plasmids extracted
  • Run a gel electrophoresis on the plasmids

 

July 11 Andrés, Pedro, & Eitan

Results from previous work session:

  • –

Advances:

  • Minipreps of pUC19 and part 3A transformants carried out.

Things left to do:

  • Prepare TAE buffer

 

July 13 Santiago

Results from previous work session:

  • –

Advances:

  • Parts luxR(‘14), luxR(‘15), luxI, pLux, aaiA, lacI, and pLac transformed using NEB chemocompetent cells.
  • 1 L TAE buffer sent to sterilization
  • 15 LB+Chlor. plates ordered from sterilization dept.

Things left to do:

  • Organize iGEM drawer
  • Check transformants, miniprep and so forth
  • Pick up TAE buffer and plates

 

July 14 Pedro

Results from previous work session:

  • All transformations were successful except for luxR(‘14)!!!! WE FINALLY HAVE SOMETHING TO WORK WITH!! :) :)

Advances:

  • LB+chlor. tubes were inoculated with transformed cells.
  • Plates with transformed cells were stored at 4ºC
  • luxR(‘14) transformants were plated onto LB+Amp. ‘14 parts appear to have ampicillin resistance cassettes in their backbones, not chloramphenicol.
  • 50% glycerol solution was prepared for use in cell conservation protocol.

Things left to do:

  • Conserve strains with all transformed parts.
  • Miniprep cells with transformed parts. Nanodrop, run gel.
  • Prepare chemocompetent cells with our Costa Rican friends’ protocol.

 

July 17 Pedro and Pablo

Results from previous work session:

  • luxR(‘14) transformants grew in LB+amp.

Advances:

  • ON cultures of all seven parts (luxR(‘15), luxR(‘14), luxI, pLux, aaiA, lacI, and pLac) inoculated.

Things left to do:

  • Minipreps, NanoDrops, gels of all 7 parts
  • Preserve our transformed cells in glycerol

 

July 18 Pedro Gabriela and Lord Eitan

Results from previous work session:

  • ON culture of luxR(‘14) did not grow. Procedure carried out with the rest of the transformants.

Advances:

  • Minipreps of luxR(‘15), luxI, pLux, aaiA, lacI, and pLac carried out.
  • NanoDrop of the plasmids carried out. All concentrations were above 100 ng/µL (5 times higher than last year!).
  • Digestions of 4 parts:
    • LuxI E-S
    • LacI X-P
    • pLac E-S
    • Aaia X-P
  • Cells from all 6 transformations (luxR(‘15), luxI, pLux, aaiA, lacI, and pLac) were preserved in glycerol at -30°C and -80°C.

Things left to do:

  • Run gel with all the plasmids
  • Plate NEB cells onto LB
  • Make liquid culture of LuxR (‘14)
  • Transform constitutive promoter part into NEB cells
  • Transform empty plasmid backbones into cells.
  • Digestions of other parts:
    • LuxR (Both) X-P
    • pLux E-S

 

July 21  Santiago

 

Advances:

  • Digestions of other parts
    • LuxR 15  X-P
    • pLux  E-S
  • E. coli transformed with pCons. The transformation was plated in LB + clor
  • NEB comercial cells were plated onto LB
  • ON culture of new E. coli DH5alpha

 

Things left to do:

  • Run gel with all the part’s digestions
  • Culture ONs of NEB comercial cells and new DH5alpha
  • Look for destination plasmids

July 22 Eitan, Diego and Eitan.  

 

Advances:

  • Electrophoresis

 

Pozo

1

2

3

4

5

6

7
 

MP

Mini. pLac

Dig. pLac

Min. LacI

Dig. LacI

Min. aaiA

Dig. aaiA



Pozo

8

9

10

11

12

13
 

Min. LuxR

Dig. LuxR

Min. pLux

Dig. pLux

Min. LuxI

Dig. LuxI

 

  • Take out from incubator to  -4°C:
    • NEB commercial cells
    • DH5alpha
  • Plate LuxR14 transformation
  • Culture ON of pCons transformation on LB + Chlor (25ug/mL)

 

Things left to do:

  • Check growth of LuxR14 transformed cells  
  • Miniprep pCons
  • Culture ONs of NEB commercial cells and DH5alpha

 

July 23  Pedro  

 

Results from previous work session:

  • Plates with transformed cells:
    • LuxR14 didn’t grow
    • pCons grew
  • pCons ON grew

 

Advances:

  • Take out from incubator to  -4°C:
    • NEB comercial cells
    • DH5alpha
  • pCons transformed cells were move from incubator to -4°C
  • LuxR14 transformed cells were plated
  • ON cultures of NEB and DH5alpha cells

 

Things left to do:

  • Check LuxR14 transformed cells growth
  • NanoDrop of pCons Miniprep
  • Prepare chemically competent cells with NEB and DH5alpha cells with Costa Rica protocol.

 

July 24 Diego, Gabriela y Pedro   

 

Results from previous work session:

  • 4 colonies grew in LuxR14 trasnformation plate

Advances:

  • NanoDrop of pCons Miniprep: 75ng/mL
  • Prepare CaCl2 100mM
  • Prepare CaCl2 100mM 15% Glycerol
  • NEB and DH5alpha were left in CaCl2 100mM

 

Things left to do:

  • Finish chemically competent cells protocol of Costa Rica with NEB and DH5alpha cells
  • Look for plasmids with RFP and resistance to chloramphenicol, kanamycin, and ampicillin.     

 

July 25 Eitan y Pedro  

 

Advances:

  • Finish chemically competent cells protocol of Costa Rica with NEB and DH5alpha cells

 

Things left to do:

  • Check chemically competent cell efficiency

 

July 27 Pablo  

 

Results from previous work session:

Advances:

  • Backbones plasmids with RFP founded
  • Transform chemically competent cells (NEB and DH5alpha) with an igem part. Costa Rica’s protocol was used.
  • Transform backbone plasmids with NEB commercial cells.
  • Plate the three backbone plasmids transformations
  • Plate igem part transformation using self-prepared chemically competent cells

 

Things left to do:

  • Check growth in transformation with self-prepared chemically competent cells
  • Culture ON of backbones plasmids transformations  

 

July 28  

 

Results from previous work session:

  • RFP Amp, RFP Kan and RFP Chlor grew.
  • iGem part transformed using self-prepared chemically competent cells didn´t grow.

Advances:

  • Culture ONs of RFP Amp, RFP Kan and RFP Chlor

 

Things left to do:

  • Minipreps of RFP Amp, RFP Kan and RFP Chlor.

 

July 29 Andrés  

 

Results from previous work session:

Advances:

  • Minipreps of RFP Amp, RFP Kan and RFP Chlor.
  • Nanodrop
    • RFP Amp    246,8 ng/mL
    • RFP Kan      93,2 ng/mL
    • RFP Chlor    79,3 ng/mL
  • Culture ON of RFP Amp plasmid

 

Things left to do:

  • Digestions of backbone plasmids
  • Transform again with self-prepared chemically competent cells

 

July 30  Andrés

 

Results from previous work session:

Advances:

  • Transformation with self-prepared chemically competent cells.
  • Miniprep of RFP Amp plasmid
    • Note: when making the plasmid digestion we also make a mistake: we add NEB Buffer 2.1 to Minipreps. Then the concentration falls to an unknown value.   
  • Culture ONs of RFP Amp, RFP Kan and RFP Chlor transformations.

 

Things left to do:

  • Minipreps of RFP Amp, RFP Kan and RFP Chlor.  

 

July 31 Andrés y Pedro   

 

Results from previous work session:

  • The RFP Amp didn’t grow, but RFP Chor and RFP Kan did.

 

Advances:

  • Culture ON of RFP Amp transformation.
  • Minipreps of RFP Amp, RFP Kan and RFP Chlor.

 

Things left to do:

  • NanoDrop of RFP Amp, RFP Kan and RFP Chlor minipreps

 

August 1 Andrés y Pedro   

 

Advances:

  • NanoDrop:
    • RFP Amp    166,5 ng/mL
    • RFP Kan      111,9 ng/mL
    • RFP Chlor    93,6 ng/mL
  • Digestions of RFP Amp, RFP Kan and RFP Chlor.

 

Things left to do

  • Ligations:
    • LuxI - LacI
    • pLac - aaiA
    • pCons - LuxR

 

August 3  

 

Advances:

  • The following ligations:
    • LuxI - LacI
    • pLac - aaiA
    • pCons - LuxR
  • Culture ONs of NEB and DH5alpha cells

 

Things left to do:

  • Look for Cytochrome + tag degradation and pClock
  • Prepare chemically competent cells using TSS Buffer
  • Plate NEB and DH5alpha cells

 

August 4   Andrés

 

Advances:

  • Prepare TSS buffer

 

Things left to do:

  • Look for Cytochrome + tag degradation and pClock
  • Prepare chemically competent cells using TSS Buffer
  • Plate NEB and DH5alpha cells

 

August 5   Gabriela y Santiago

 

Results from previous work session:

Advances:

  • Electrophoresis

 

Pozo

1

2

3

4

5

6

7
 

MP

Mini. RFP Amp

Lig. LacI-LuxI

Lig. pCons- LuxR

Lig. pLac-aaiA

Mini. pLac

Mini. aaiA
  • Filtrate TSS
  • Prepare SOB

 

Things left to do:

  • Prepare chemically competent cells
  • Plate NEB and DH5alpha cells
  • Adjust SOB pH (approximately 12.5mL of NaOH 1M)
  • Add glucose to SOB

August 6  Andrés    

 

Advances:

  • Adjust SOB pH (approximately 12.5mL of NaOH 1M)
  • Add glucose to SOB
  • Filtrate SOC
    • Note: the chemically competent cells couldn’t be prepared because the OD reach 0.9 to 1.0
  • Plate transformated parts: aaiA, LuxI, pLac, LacI, pLux and LuxR15
  • Plate NEB and DH5alpha cells



Things left to do:

  • Plate the remaining parts
  • Prepare chemically competent cells
  • Culture ONs of ligations

 

August 10 Pablo   

 

Results from previous work session:

  • Had grown red and white colonies to each transformation (LuxI - LacI, pLac - aaiA & pCons - LuxR)

 

Advances:

  • Culture ONs of LuxI - LacI, pLac - aaiA & pCons - LuxR (white colonies) on LB + Amp

 

Things left to do:

  • Prepare chemically competent cells
  • Check bacteria growth in ONs

 

August 12 Eitan    

 

Results from previous work session:

  • Some ONs grew red.

 

Advances:

  • Culture ONs of LuxI - LacI, pLac - aaiA & pCons - LuxR (white colonies) on LB + Amp
  • Plate:

LB + Chlor

    • pLux
    • LuxI
    • pLac
    • aaiA
    • LuxR15
    • LacI
    • LuxR14(Amp)

 

LB

    • DH5alpha cells
    • NEB Cells

 

Things left to do:

  • ON to prepare chemically competent cells
  • Minipreps of ligations (LuxI - LacI, pLac - aaiA & pCons - LuxR)

 

August  13  Diego y Pedro  

 

Advances:

  • Minipreps of
    • LacI - LuxI
    • pCons - LuxR
    • pLac - aaiA
  • Culture ONs of DH5alpha y NEB

 

Things left to do:

  • NanoDrop of
    • LacI - LuxI
    • pCons - LuxR
    • pLac - aaiA

 

August 14 Gabriela   

 

Results from previous work session:

  • aaiA and DH5alpha plates were useless. The rest is ok and was moved to -4°C.

 

Advances:

  • Plasmid concentration measurements using NanoDrop of these ligations:
    • LacI - LuxI        63.4  ng/uL
    • pCons - LuxR   216.6 ng/uL
    • pLac - aaiA        34.5 ng/uL

 

Things left to do:

  • Plate aaiA on LB + Chlor and DH5alpha on LB
  • Digestions of first ligations (LuxI - LacI, pLac - aaiA & pCons - LuxR)

 

August 18 Pedro   

 

Advances:

  • Digestions of:
    • LacI - LuxI        E-S
    • pCons - LuxR   E-S
    • pLac - aaiA       X-P
    • pLux                X-P



Things left to do:

  • Plate aaiA on LB + Chlor and DH5alpha on LB
  • Ligations:
    • pCons-LuxR + pLux
    • LuxI-LacI + pLac-aaiA

 

August 21 Pedro   

 

Advances:

  • Ligations:
    • pCons-LuxR + pLux
    • LuxI-LacI + pLac-aaiA

Note: destination plasmid RFP Chlor

 

Things left to do:

  • Plate aaiA on LB + Chlor and DH5alpha on LB
  • Prepare chemically competent cells
  • Transform with electroporation and chemical transformation protocol

 

August 24  Eitan   

 

Advances:

  • Plate of NEB and DH5alpha both on LB
  • Culture ONs of NEB cells and DH5alpha both on LB

 

Things left to do:

  • Prepare chemically competent cells
  • Transform with electroporation and chemical transformation protocol

 

August 25 Andrés   

 

Results from previous work session:

  • Plates and ONs of NEB cells and DH5alpha grew

 

Advances:

  • Prepare chemically competent cells using SOC and LB media

 

Things left to do:

  • Transform self-prepared chemically competent cells using both the chemical transformation and electroporation protocols

 

August 26 Gabriela    

 

Advances:

  • Transform self-prepared chemically competent cells using both the chemical transformation and electroporation protocols

 

Things left to do:

  • Check growth on transformations using self-prepared competent cells and chemical and electroporation protocols

 

August 31 Pablo y Pedro   

 

Results from previous work session:

  • Growth of transformation with self-prepared competent cells:
    • Electroporation and SOC media: didn’t grow
    • Electroporation and LB media: didn’t grow
    • Chemical transformation protocol and SOC media: didn’t grow.
    • Chemical transformation protocol and LB media: didn’t grow.

Advances:

  • Electrophoresis

 

Well

1

2

3

4

5

6

7

8
 

Weight ladder

lig. pCons-LuxR + pLux

lig. LuxI-LacI + pLac-aaiA

dig. pCons-LuxR

dig. pLux

dig. LuxI-LacI

dig. pLac-aaiA

RFP Chlor plasmid




Things left to do:

  • Check each step (MiniPrep, digestions and ligations) because  the gel showed that ultimate ligations are wrong and problems with the digestions of ligation components.Check it with electrophoresis gels.  

 

September 3    

 

Results from previous work session:

  • The gel showed that ultimate ligations are wrong and problems with the digestions of ligation components

 

Advances:

  • Scheme of our procedures (minipreps, digestions and ligations) with the date when were made.

 

Things left to do:

  • Check procedure steps with electrophoresis gel.
  • Wash self-prepared competent cells from salts, to electroporation.

 

September 5 Pedro   

 

Advances:

  • Electrophoresis



Well

1

2

3

4

5

6

7
 

Molecular Weight Ladder

Dig. pCons E-S

Dig. LuxR15 X-P

Dig. pLac E-S

Dig. LuxI E-S

Dig. PLux E-S

Dig. LacI X-P



Well

8

9

10

11

12

13

14

15
 

empty

Molecular Weight Ladder

Dig. RFP Chlor E-P

Dig. RFP Kan E-P

Dig. RFP Amp E-P

Lig. RFP Amp  pLac- aaiA

Lig. RFP Amp

pCons-LuxR

Lig. RFP Amp LacI-LuxI

 

Note: the gel didn’t run. It could be explain by a low quality TAE buffer.

 

Things left to do:

  • Repeat electrophoresis

 

September 10 Pedro   

 

Results from previous work session:

  • The gel didn’t run. It could be explain by a low quality TAE buffer.

 

Advances:

  • Electrophoresis
  • Dig. LuxR15 X-P

 

Well

1

2

3

4

5

6

7
 

Molecular Weight Ladder

Dig. pCons E-S

Dig. LuxR15 X-P

Dig. pLac E-S

Dig. LuxI E-S

Dig. PLux E-S

Dig. LacI X-P



Well

8

9

10

11

12

13

14

15
 

Dig. aaiA X-P

Molecular Weight Ladder

Mini. RFP Amp

Dig. RFP Amp E-P

Mini. RFP Kan

Dig. RFP Kan E-P

Mini. RFP Chlor

Dig. RFP Chlor E-P

 

Note: The electrophoresis ran 85 minutes, not 60 as was planned. Just three band were observed in Molecular weight ladder wells and a single ban in another well.

 

Things left to do:

  • Repeat electrophoresis

 

September 11   

 

Results from previous work session:

  • The electrophoresis ran 85 minutes, not 60 as was planned. Just three band were observed in Molecular weight ladder wells and a single ban in another well.

 

Advances:

  • Electrophoresis

 

Well

1

2

3

4

5

6

7
 

Molecular Weight Ladder

Dig. pCons E-S

Mini. RFP Amp

Dig. LuxR15 X-P

Dig. RFP Amp  E-P

Dig. pLac E-S

Dig. LuxI E-S



Well

8

9

10

11

12

13

14

15
 

Dig. LuxI E-S

Molecular Weight Ladder

Dig. RFP Kan E-P

Dig. PLux E-S

Mini. RFP Chlor

Dig. LacI X-P

Dig. RFP Chlor E-P

Dig. aaiA X-P



               
               

 

                 
                 

 

Note: were observed bands in molecular weight ladder wells and in well 4. just one band was observed in well 4. It was near to the well so we think it is the plasmid.   








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