Team:CityColSanFrancisco/Notebook/Protocols

Media Prep:

LB (Lauria Broth) 1 Liter:

Bacto-tryptone                    10g

Bacto-yeast extract              5g

NaCl                                           10g

For agar plates add:

Bacto-agar                              15g

YP (Yeast Peptone) 1 Liter:

Yeast extract                          10g

Bacto-peptone                     20g

Glucose                                                    20g

For agar plates add:

Bacto-agar                              15g

 

FEM (anaerobic media) 1 Liter:

*PLEASE use all appropriate PPE for the preparation of this media.  Aerosols are harmful!

All proponents for media are weighed on a digital balance.  Approximately 75-80% of the distilled water is added to the beaker on a stir plate.  Once all ingredients have been added to the dH₂O and homogenized for a few minutes (no solid is seen) the contents are added to a graduated cylinder and brought to volume with dH₂O.  The media is then transferred back into the beaker and put back on the stir plate for final homogenization.  After thorough mixing, the media is either transferred into a large autoclavable bottle or aliquoted for liquid culture in Morton closure glass tubes.  It is important to leave capped closures partially opened, as the increase of heat and pressure creates gas that will expand beyond the volume of the container.  Also, it is important to remove the liquid from the autoclave once the containers have cooled to a reasonable temperature.  The caps are then tightened to avoid contamination and evaporation.  Leaving the liquid in the autoclave promotes unnecessary evaporation and concentration of the liquid media. 

When making media to pour plates, the agar is added last.  The stir bar should remain in the media for the autoclave cycle and the media should be “homogenized” after the container is at an appropriate temperature.  The agar will not incorporate into the media until after autoclaving as its melting point is quite high; trying to incorporate it into the media pre-autoclave is futile.  Label all plates before pouring them!!  Plates must be sterilely poured before the agar begins to congeal.

Any antibiotics that are to be added to media (either liquid or for plates) must be added after autoclaving.  They do not survive this process, and are rendered useless.  If antibiotics were added before, they must be added again afterwards, and only when the liquid is cooled significantly (50-55°C).

 

Cell Culture:

P. aeurigonsa grown in LB - 37°C

R. palistrus (both strains TIE-1, TIE-3) grown in YP - 30°C

E. coli grown in LB - 37°C

R. ferrireducins grown aerobically in both LB and YP, but anaerobically in FEM -

S. oneidensis  (all three strains: WT, MTRB, OMCB) grown in LB - 37°C

 

After the media is prepared and autoclaved in liquid cycle, liquid cultures are inoculated from the culture slants received from various labs.

This begins the cycle of creating plates from liquid cultures, then selecting a single colony from a plate to inoculate another liquid culture.  Every 48 hours, plates were created from liquid cultures, then after another 48 hours, liquid cultures were inoculated again. 

Liquid cultures were also used to create a frozen cell bank.  Glycerol (50-90% - depending on what was available, and that was sterile) was used for preserving the culture by preventing crystallization of the cell walls.  The liquid culture was added to the Cryo-tube first, and then glycerol added on top.  After mixing, they were placed in a -80°C freezer.

 

Plasmid Digestion:

Two plasmids were digested:  pSB1A3 (high copy plasmid) – 2156bp (small fragment – 22bp, large             fragment – 2134bp)

                                                           Bba_J63009 (low copy plasmid) – 2098bp (sm fragment – 21bp, lg fragment – 2077bp)

Plasmids were purified and eluted in optimized buffer for a final concentration of 150ng/ μL. 

Protocols from iGEM were followed.  The plasmids were cut with EcoRI and SpeI.  Measurements are as follows:

1μL EcoRI

1μL SepI

5μL Buffer 4                                                        Because 4 reactions were done for each plasmid type,                     

0.5μL BSA                                                            this recipe should be multiplied by 4, then evenly aliquoted 

5μL of plasmid DNA                                         into 4 tubes for digestion.  (TV: 45μL per reaction)

37.5μL dH₂O

The DNA is added last to the mix.  Each tube was incubated at 37°C for 45 minutes.  This should give complete digestion.  The tubes are then put into an 80°C heat block for about 10 minutes to deactivate the enzymes, and stop the digestion process.  Portions of the product are run through gel electrophoresis to determine if digestion has happened, and is complete.

 

PCR Reactions:

Several reactions have been done.  Traditional and Colony PCR were the most useful to this project.  Primers were designed based on the OprB gene (1340bp) found in P. aeurginosa.  The amount needed for each reaction is based on final concentration, and total volume.  A spreadsheet was created to help with calculations, depending on the total volume for each reaction done.  Reaction volume was also amended based on the amount of genomic DNA available.  Colony PCR uses a single colony from a plate, that is added individually to each of the reaction tubes just before going into the thermocycler. 

Parameters for cycling were changed depending on the reaction, primers used, and as a troubleshooting tool to optimize the reactions.