Team:NYMU-Taipei/Experiments/Protocols

Cloning
Our cloning cycle consists of two days: a light day (not too much to do) and a heavy day (lots to do).
 * 1)  (light day)
 * 2) Plasmid extraction and digestion (first part of heavy day).
 * 3) Gel extraction/Purification, ligation, and transformation (second part of heavy day).

Colony selecting and checking

 * 1) Preparation
 * 2) * Circle the selected n colonies on the plate.
 * 3) * Prepare a checking plate (divide the plate into n sections)
 * 4) * Prepare enough 4ml LB in 10ml tubes (depending on how many cultures you want).
 * 5) * Prepare at least n+2 PCR tubes (2=positive+negative)
 * 6) Action
 * 7) For each colony: pick the selected colony off (using a tip or toothpick) then:
 * 8) * Dip it in the PCR solution.
 * 9) * Dip it in the liquid (optional).
 * 10) * Streak it on the checking plate.
 * 11) End of action
 * 12) * Put the checking plate and the liquid cultures into the incubator for 12-16hrs.
 * 13) * Put the PCR tubes in the PCR machine and run.
 * 14) When the PCR is done, run it on gel electrophoresis then
 * 15) * Mark the colonies (on the checking plate) which are correct and incorrect.

Plasmid extraction and digestion

 * For plasmid extraction, we use Viogen's miniprep kit. This usually takes around an hour.
 * For digestion using restriction enzymes, we follow the protocol provided (we use different brands). Then we put it at 37C for various amounts of time ranging from 1/2hr to a few hours, the average being around 2hrs.

Gel extraction/purification, ligation, and transformation

 * Depending on the digestion, we either perform gel extraction or just directly purify it.
 * For that, we use [Someone's] Gel extraction and PCR purification kits respectively.
 * Ligation is performed following the provided by the manufacturer (again, we use different brands). Usually we leave it in the cupboard (about 22C-25C) for about 17mins before doing transformation. Depending on the difficulty of the ligation, we also ligate overnight at 4C.
 * We transform using [someone's] 5 minute transformation protocol, then incubated at 37C for 12-16hrs. Depending on the origins of the plasmid we're transforming, we use different amounts of competent cells. Amounts:
 * 25uL: is usually sufficient.
 * 16-20uL: For those that grow a lot of colonies.
 * 33uL: For transforming straight from the biobrick plates.
 * 50uL: For transforming biobricks that are difficult to transform.

"Magic" Colony PCR
Magic PCR protocol:

Ideal for 19 or 23 PCR tubes.
 * For 19 tubes -> take 24.5ul/tube (24.5ul wasted on tips)
 * For 23 tubes -> take 20ul/tube (30ul wasted on tips)

23 tubes (21 colonies + positive and negative control) is ideal since it fits snugly on a 25-well gel, and also one plate can be divided into 24.

Reporting Assay
Day -2: Day -1: Day 0:
 * 1) Plates streaked to produce single colonies.
 * 2) Plates grown overnight (12-16hrs) at 37C.
 * 1) For each part to test, three 3ml cultures of LB and ampicillin (10mM) were inoculated with single colonies from the freshly streaked plates (three biological replicates) in a 10ml tube.
 * 2) Cultures were grown overnight (12-16hrs) at 37C shaking at 180-200rpm.
 * 1) The cultures were diluted to 0.0325 OD600 into 3-5ml (usually 3ml) and grown for 1hr at 37C shaking at 180-200rpm (keep the shaking speed consistant throughout.)
 * 2) For every 1hr for 5 or 6 hours, do:
 * 3) Take out the liquid cultures
 * 4) For all liquid cultures for, do:
 * 5) Extract 250ml into a cuvette that is able to measure the OD600 with only 250ml.
 * 6) After measuring the OD600, extract 200 and put it into a well on a black 96-well plate.
 * 7) Put the liquid cultures back into the incubator and let the continue growing (note the time taken from taking out the liquid culture to putting it back. This time should be kept as consistant as possible).
 * 8) Measure the Fluorescence.

<!--#Three 5 ml cultures of supplemented M9 medium and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of MG1655 containing BBa_T9002. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of MG1655 containing a BBa_T9002 mutant (T9002m) lacking a GFP expression device described in the stability section.
 * 1) Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
 * 2) Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.
 * 3) Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner).
 * 4) 2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone (3OC6HSL), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of 3OC6HSL.  Three wells were each filled with 200 µl of medium to measure the absorbance background.  Three further wells were each filled with 200 µl of the BBa_T9002 mutant culture to measure fluorescent background.
 * 5) The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s.  Approximately 6 min elapsed between beginning addition of 3OC6HSL to the wells and the first plate reader measurement.  3OC6HSL was added in order of increasing concentration to minimize GFP synthesis during plate loading.  Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).
 * 6) We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate.
 * 7) We processed the data to compute the PoPS output from BBa_F2620 as described on the Data analysis page. The data for each colony tested was averaged across the three replicate wells.  The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3.  Following an initial transient response, device output reached an approximate steady state.
 * 8) The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples.  The cyan shaded region represents the range of the nine independent samples.
 * 9) To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R2=0.99).  In the equation (shown below), Pout is the PoPS per cell output of BBa_F2620, Pmax is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.
 * 10) To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate 3OC6HSL concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data.  The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate 3OC6HSL concentrations -->.