Team:Reading/Lab Work

Lab Book

Here we present our lab book: notes, results and any changes to protocols. These notes are mostly unaltered, presented as they were taken over the course of the lab based aspect of the project. Any alterations were to make them more legible for the web. We hope these brief notes offer insight into the daily goings of our lab team. In addition to this all protocols we used are presented on their own page. The lab book is presented in chronological order. All dates are English format ie. DD/MM/YYYY.

14/07/2014

Set up initial cultures of PCC 7942 according to protocol

Measured OD₇₅₀ in order to quantify growth of cyanobacteria. OD measurement preformed according to protocol.

15/07/2014

Following acquisition of DNA ligase we begin BioBrick assembly using protocol available on protocol page.

• Before assembling the BioBricks we checked the concentration of DNA which were attained from miniprep. Protocol for checking DNA concentration suing Nanodrop is available in protocols section.

  Person	Measurement	260nm	280nm	230nm	DNAng/ul
  SH	1	0.641	0.356	-	32.0
  HC	1	0.465	0.261	0.300	23.3
  	2	0.500	0.253	0.294	25.0
  MS	1	0.473	0.240	0.367	23.0
  	2	0.460	0.265	0.340	23.6
  OS	1	0.221	0.120	0.287	11.0
  	2	0.185	0.122	0.307	8.2

• Each sample was measured twice to obtain a more accurate reading.

17/07/2014

Completed BioBrick assembly by transforming competent NEB 5-alpha E. coli. Transformation was preformed according to protocol

• Bacteria not plated stored at 4℃

Prepared selection plates containing ampicillin ready for plating according to protocol

18/07/2014

Checked plates from Biobrick assembly the previous day.

• No growth seen on an any plates after 18 hours.

• Transformed bacteria replated onto regular agar plates and incubated at 37 ℃ to confirm viability of cells.

21/07/2014

OD₇₅₀ of 7942 cultures from 14/07/2014 measured = 0.080.

• This media was subcultured using 2 ml of culture into 100 ml of BG11 media into a new 250 ml conical flask.

22/07/2014

6803 arrived from Howe lab in Cambridge. We also received 3 plasmids for transformation. We have received both wild type (WT) and mutant (terminal oxidase) strains.

• Started new liquid cultures of 6803 by using 3 loops of bacteria to inoculate 200 ml of BG-11 agar

• Created 2 streak plates, one WT and one mutant, on solid BG-11 plates. Protocol for making BG-11 plates is available in protocol section.

• Spun down plasmids from Howe lab and stored at -20℃.

Checked OD₇₅₀ of 7942 cultures = 0.17.

• Subcultured 7942 with 2 ml from our growing culture and stored it in warm room under light on shaker at 55 rpm.

Preformed Gram stain of 6803 using standard protocol and visualised cells under a light microscope.

23/07/2014

Using a new batch of NEB 5-alpha E. coli we retried BioBrick from 17/07/2014.

• This time we stored thawed cell on ice rather than in a water bath. Water bath as at ~44℃ rather than the recommended 42℃.

• Plated 7 plates of LB agar or Amp LB agar.

Checked cultures. Solid 6083 WT showed growth while 7942 subculture did not.

25/07/2014

Checked OD₇₅₀ of original 7942 and 6803. Data added to online database.

class="title">28/07/2014

Installed new white light.

Checked lux of lamp above the incubator.

31/07/2014

Made a fuel cell using 7942 from a culture from 14/07/2014.

• Voltage was measured at 0.2 V however no current was measured

01/08/2014

Started new cultures of the WT and mutant 6803 from 22/07/2014 cultures by placing 1 ml of culture into 100 ml of BG-11 media.

• Measured OD of these new cultures:
  • Original WT = 1.707 Subcultured = 0.019
  • Original MT = 1.187 Subcultured = 0.025

04/08/2014

Transformed E. coli using 1 plasmid from Cambridge and plated onto amp LB agar plates. Incubated overnight at 37℃.

Send 60% glycerol for autoclaving.

05/08/2014

Checked plates from previous days transformations. Took grown colonies and inoculated LB broth in 4 bijous. These were placed on a shaker at 200 rpm 37℃. Could not add antibiotic (Amp) to the broth as it was not available.

06/08/2014

Made glycerol sock of 6803 according to protocol

• Placed in box in (A1-A7) in -80℃ in G49

Preformed MiniPrep of plasmid using LB E. coli. Tested concentration of DNA on nanodrop = 35 ul/ml

07/08/2014

Made kanamycin plates

• 1.2 g of agar added to 200 ml sterile nano pure water
• Added 2 ml of 50 mg/ml kanamycin

08/08/2014

Checked OD of cultures

Pored BG-11 plates

• Used 30 ug/ml of kanamycin rather than 50 ug/ml as stated in the protocol

Diluted 6803 WT cultures from 22/07 and 6803 MT from 22/07 to get and OD required for transformation

14/08/2014

Growing cyanobacteria on carbon fibre

• Soaked carbon fibre cut into disk in 6 ml of BG-11 in a petri dish
• Added 3 ml of WT 6803 (subc. 01/08/2014) to the surface of carbon fibre
• Left to grow on floor under light at 28℃

Preformed mini prep according to protocol and checked concentration on nanodrop according to protocol. Plasmid concentrations = 22.1 ng/ul and 29.7 ng/ml

15/08/2014

Diluted 10ul ampicillin (50 mg/ml) in 10 ml LB to get 50 ug/ml. Send to be autoclaved ready for making plates.

18/08/2014

Checked OD of cultures

Created new cultures of both the WT and the MT from cultures from 06/08/2014 and 01/08/2014

Made BG-11 agar

• 7.55 g agar to 100 ml BG-11

19/08/2014

Sent NaHCO3 and BG-11 to be sterilised

20/08/2014

Made BG-11 plates

• Used sterilised BG-11 agar from the 18th

• added 400ml BG-11 warmed to ~50C

• Gave 500ml BG-11 agar

• used ~100ml for normal BG-11 plates

• used ~200ml for BG-11 + 50ug/ml Kan (200ul of 50mg/ml Kan)

• used ~200ml for BG-11 + 30ug/ml Kan (120ul of 50mg/ml Kan)

• gives 6x BG-11, 10x BG-11 kan 50ug/ml,

26/08/2014

Made 0.6% BG-11 plates as described in protocol

• Used 1.2 g for 200 ml BG-11 agar

03/09/2014

Checked ODs

Re-started transformation protocol - innoculating 2 OD ~2

05/09/2014

Preformed miniprep on E.coli after 2 days of growth. Results unsatisfactory. Left E.coli in incubator over the weekend

Set up liquid cultures of E.coli containing flavin biobrick in preparation for miniprep

08/09/2014

Collected and preformed serial dilution of pond water. 50 ul of 10-1 to 10-5 was plated onto ager plates. Left at 20℃

Completed miniprep of flavin containing E.coli

• Measured DNA concentration on nano drop
  • 1.1 - 204.3
  • 1.2 - 93.6
  • 2.1 - 159.9
  • 2.2 - 43.1
• Extracted flavin biobrick left in freezer at -20

09/09/2014

Ferricyanide assay at 11 am

• OD750 light sample - 3.89
• OD680 light sample - 5.00
• OD420 light sample - 8.41
• OD750 dark sample - 4.17
• OD680 dark sample - 5.43
• OD420 dark sample - 8.97

  --------------------------------------------------

• OD750 light sample - 0.18
• OD680 light sample - 0.16
• OD420 light sample - 1.05
• OD750 dark sample - 0.12
• OD680 dark sample - 0.13
• OD420 dark sample - 0.98

10/09/2014

Ferricyanide assay at 10:30 am

• OD750 light sample - 4.16
• OD680 light sample - 5.51
• OD420 light sample - 8.99
• OD750 dark sample - 3.82
• OD680 dark sample - 5.26
• OD420 dark sample - 8.84

  --------------------------------------------------

• OD750 light sample - 0.28
• OD680 light sample - 0.31
• OD420 light sample - 1.27
• OD750 dark sample - 0.13
• OD680 dark sample - 0.17
• OD420 dark sample - 1.02

Pored BG-11 plates according to protocol

18/09/2014

Transformed with parts from the registry (Plates location - name):

• 11A - BBa_K516132
• 5I - BBa_K608008
• 5M - BBa_K608011
• 23O - J04450 x 2
• Plate 4 6B - J04450
• Used invitrogen procedure for 3 and iGEM procedure from 3
• 1 ul if DNA from the BioBrick distribution was used for each transformation, except for one version of 23O which used 2 ul.

23/09/2014

Transformation of 6803 planned according to following table:

Number	1	2	3	4	5	6
Amount	1ml	1ml	1.5ml	1.5ml	1ml	1.5ml
Temp. incubated	28 overnight	28 overnight	28 overnight	28 overnight	34 at 1.45pm removed at 5.05pm and place at 28 overnight	34 at 1.45pm removed at 5.05pm and place at 28 overnight
Light while incubated?	yes	no	yes	no	no	no

• Final amount will be about 120 ul in each tube. Plate half on BG-11 plates and half on Kan 50 plates. Do not stack plates, place with agar facing up.
• OD of 6803 culture for transformation is 0.435
• All washes done by resuspending in 1 ml of fresh BG-11 (regardless of the initial volume of culture)
• Used plasmid DNA at a concentration of >270 ng/ul
• Added 8 ul of plasmid to each tube to give >2 ug plasmid DNA for each tube
• Tubes were placed in light and temperature condition as outlines in the table. Tubes 1-4 were put straight into the warm room and placed on the shaker, while tubes 5 and 6 were put in a 34℃ water bath, unshaken, for 3:20:00 and were then transferred to the warm room with the rest of the tubes.
• We forgot to shake the tubes every 1-2 hours, so may see reduced transformation efficiency because of this. Left them overnight, rather than for just 4-5 hours, this may also affect transformation efficacy. Tested growth of pond wa8ter
• 3 replicates of each point water bottle, A and B, were used to test cyanobacteria growth in UV-sterilised pond water, giving cultures A1-3 and B1-3.
• OD750 of 1.4 was recorded for the cultures used for inoculating pond water. Spectrophotometer was blanked with BG-11.
• ODs were checked for pond water inoculated with 10 ml of the culture measured above. in this case the spectrophotometer was blanked with pond water.
• About 150 ml of pond water was used in each case.
• They were inoculated into 250 ml conical flasks under sterile conditions and placed in a 75rpm shaker at 27℃ under light.
• Results were as follows: taken at time 0 (5pm on 23/09/2014)
  • A1: 0.170
  • A2: 0.124
  • A3: 0.145

  • B1: 0.113
  • B2: 0.137
  • B3: 0.115

24/09/2014

Did stuff with biobrick. It didn't work. It never works.

25/09/2014

Pond water OD750. Taken at 10:30

• A1: 0.176
• A2: 0.196
• A3: 0.181

• B1: 0.215
• B2: 0.235
• B3: 0.186

26/09/2014

Did stuff with biobrick. It didn't work. It never works.

29/09/2014

Measurement of pond water cultures

• A1: 0.405
• A2: 0.438
• A3: 0.400

• B1: 0.643
• B2: 0.757
• B3: 0.597

Facebook

Twitter

rusynbioigem@gmail.com