Difference between revisions of "Team:Glasgow/Project/Overview/RBS"

Charlotte Flynn - Carried out cloning of devices, measurements of devices and completed the relevant forms and content of wiki page.

Sean Collums - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page.
Vilija Lomeikaite - Set up overnight cultures of the devices
Ye Yang - Designed and formatted the Interlab wiki page
Andrey Filipov - Carried out the calibration measurements for the spectrophotometer
James Provan - Sent cloned devices for sequencing

The cloning of devices was carried out from the 17 – 21st of August. Measurements of the devices were carried out from the 24th– 28th of August. Detailed lab book.

Model and manufacturer:
o Incubator – 2cm shaking diameter
o Spectrophotometer – Used to measure absorbance at 600nm of each sample.
o Typhoon FLA 9500 - GE Healthcare Life Sciences. Wavelength used to excite cells - 475nm. Filter/channel used to capture the light emission from the cells - Filter BPB1 (530DF20).

In order to calibrate the spectrophotometer a dilution series of 1-100% of DH5 alpha cells was carried out and the A600 of each sample was measured (Figure 1).

A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 1) and a calibration curve (Figure 3) carried out to calibrate the Typhoon. Fluorescent proteins derived from voltage (LOV) domains are smaller and more efficient under anaerobic conditions than green fluorescent proteins (GFP) (Buckley et, al. 2015). iLOV, an improved LOV flavoprotein, was originally engineered as a reporter for viral infection from phototropin, the blue light receptor. We used phiLOV which is a photostable version of the iLOV fluorescence reporter.

The devices, as shown in Table 2, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3 (Figure 4). Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.

Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the he broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates.

The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O.

The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate.

A negative control for background cell fluorescence was included as cells containing the device R0040 but without a promoter, to mimic burden of the promoter. A positive control for GFP fluorescence was included as cells containing the device I20270, a GFP part with the promoter J23151. PBS was used to control for media-only background. In addition in order to obtain absolute values for fluorescence, set standards of FAM oligo were also measured.

We used a 6-FAM (6-carboxyfluorescein) labelled oligonucleotide to standardise our fluorescent results. This allowed us to express our GFP levels as equivalent amounts of 6-FAM. 6-carboxyfluorescein is the most commonly used fluorescent dye for labelling oligonucleotides, and therefore should be readily available to most iGEM teams. 6-FAM labelled oligonucleotides can be quantitated by measuring the UV absorbance at 260 nm (measuring the DNA concentration). 6-FAM has similar fluorescent properties to eGFP (excitation peak at 492 nm and an emission maximum of 517 nm for 6-FAM compared to 488 nm excitation and 508 nm emission for E0040 GFP mut3b).

A dilution series of FAM labelled oligonucleotide was measured (Figure 5) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 6) has a line gradient of 4.79x10^6. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.

The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).


The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).

1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279).
2. The average absorbance of control E.coli cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475).
3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6).
4. Dividing these values by the conversion factor as determined from the FAM oligo dilutions (479,000) gives the absolute fluorescence equivalent to pmol of FAM oligo per A600 of cells (Table 6).

We attempted to estimate the absolute number of GFP molecules per cell (Table 7) using our phiLOV results and some simplifying assumptions.
In order to estimate the absolute number of GFP molecules per cell the following calculations were carried out:

ilov stock = 1.35 mg/ml = 1.35 g/l

MW = approx. 150x110 = 16500

ilov stock = 82uM

Avogadro’s number = 6.02x10^23
⇒ Diluted stock 2 fold and used 100 ul in well = 1/20000 litre = 4.1 nmoles
⇒ 4.1 nmoles of phiLOV gave a reading of 490,000,000

J23101:I13504

⇒ 200ul cells at A600 of 1.0 with J23101 promoter gave fluorescence reading of average 1,000,000,000.

• So 200 ul cells equivalent to 4.1 *1000/490 = 8.4 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

• So 8.4 nmoles iLOV gives equivalent fluorescence to 8.4/11.5 = 0.73 nmoles of GFP per 200 ul cells
• So 200 ul cells contains 0.73x10^-9 x (Avogadro’ s number 6.02x10^23) = 4.4x 10^14 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

• So 1 cell contains 4.4 x 10^14 / 4 x10^8 = 1 million copies of GFP

⇒ 1 million molecules of GFP = (1000000/6.02x10^23) = 1.7x10^-18 moles GFP

• 27,000 x 1.7 x 10^-18 = 4.7 x 10^-14 g = 47 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

• So approximately half of all cellular protein is GFP

J23106:I13504

⇒ 200ul cells at A600 of 1.0 with J23106 promoter gave fluorescence reading of average 250,000,000.

• So 200 ul cells equivalent to 4.1 *25/49 = 2.09 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

• so 0.209 nmoles iLOV gives equivalent fluorescence to 2.09/11.5 = 0.181 nmoles of GFP per 200 ul cells so 200 ul cells contains 0.181x10^-9 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^14 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

• So 1 cell contains 1.08x10^14 / 4 x10^8 = 272,405 = 270,000 copies of GFP

⇒ 270,000 molecules of GFP = (270,000/6.02x10^23) = 4.48x10^-19 moles GFP

• 27,000 x 4.48x10^-19 = 1.2 x 10^-14 g = 12 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

• so 12% of cellular protein is GFP

J23117:I13504

⇒ 200ul cells at A600 of 1.0 with J23117 promoter gave fluorescence reading of average 2,500,000

• So 200 ul cells equivalent to 4.1 *25/4900 = 0.0206 nmoles iLOV

⇒ GFP approx. 11.5 x brighter than iLOV per mol

• so 0.0206 nmoles iLOV gives equivalent fluorescence to 0.0206/11.5 = 1.79x10^-3 nmoles of GFP per 200 ul cells
• so 200 ul cells contains 1.79x10^-12 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^12 molecules

⇒ Fluorescence was quoted for cells at OD600 = 1.0
⇒ 1 OD600 of TOP10 = 4x10^8 cells per 200 ul

• So 1 cell contains 1.08 x 10^12 / 4 x10^8 = 2,700 copies of GFP

⇒ 1 million molecules of GFP = (2,700/6.02x10^23) = 4.48x10^-21 moles GFP

• 27,000 x 4.48x10^-21 = 1.2 x 10^-16 g = 0.12 femto grams

⇒ typical total protein content of bacterial cell = 100 femto grams

• so 1.2x10^-3 % of all cellular protein is GFP