Team:Reading/Lab Work

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University of Reading

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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.

15th July

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.

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

Took OD of samples

Created new subculture of 6803 WT from the 6803 WT from 28/07/2014.

Placed the original 6803 WT from 28/07/2014 on the windowsill to test if the OD decreases or increased in natural light

Fuel cell measuring 0.14V and 5mA

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

Preformed transformation using WT 6803 and plasmid send to us by Cambridge.

Transformation carried out according to protocol
OD was 0.9

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

NaHCO3 stock

made up 500 ml of 10mM NaHCO3 stock solution and autoclave
added 0.42005 g to 500 ml nano pure water

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,

NaHCO3 cultures

Added NaHCO3 to MT and WT cultures from 18/08/2014

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

Checked temperature of fuel cell - operating at room temperature (22℃)

Preformed ferricyanide assay at 4 pm

OD750 light sample - 4.82
0D680 light sample - 6.19
0D420 light sample - 4.30
OD750 dark sample - 3.69
OD680 dark sample - 4.72
OD420 dark sample - 2.25
--------------------------------------------------
OD750 light sample - 0.14
OD680 light sample - 0.03
OD420 light sample - 1.1
OD750 dark sample - 0.05
OD680 dark sample - 0.03
OD420 dark sample - 1.08

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