Team:UCL/Labbook

June

Week 1-3

No lab work

Week 4

26th June - We are given safety training in the Advanced Centre for Biochemical Engineering in all relevant laboratories, as well as general procedures in case of emergency.

July

Week 5-6

No lab work

Week 7

Bacterial Lab

15th July - The team is introduced to the laboratories which will be used during the summer for both bacterial and mammalian experiments.

16th July - 5X M9 salts [link to protocol], minimal agar [link to protocol], 1.4% molten agar solution [link to protocol] and 0.1M CaCl2/15% glycerol [link to protocol] were prepared for the generation of competent cells. Minimal agar plates were poured and streaked [link to streaking protocol] with W3110 Escherichia coli cells and left overnight to incubate at 37C.

17th July - Very little colony growth was observed from W3110 E.coli streaked plates. Plates were therefore left to incubate for a further 17 hours.

18th July - Sufficient colony growth allowed for the selection of a single colony from each plate. This was then inoculated in 5ul LB media [link to LB recipe] + 100ul 1M MgSO4 and left to incu-shake overnight at 37C.

19th July - Cultures re-suspended in new LB media and 100µl aliquots placed into individual eppendorf tubes for placement at -80C.

Mammalian Lab

17th July - Mammalian cell culture and maintenance [link to mammalian protocol] training by Mrs. Ludmilla Ruban. Passaged primary MEF (mouse embryonic fibroblast) cells. Passage 3.

18th July - MEF passage 4

19th July - MEF passage 5

Week 8

Bacterial Lab

22nd July -Transformation [link to transformation protocol] of our competent cells with plasmid YB3110 was carried out.

23rd July - No colony growth was observed on Ampicillin plates [link to ampicillin plate protocol] indicating no plasmid uptake. Transformation was repeated with YB3110.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

24th July - No colony growth from main experiment indicating no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

25th July - No colony growth and hence no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

26th July - Once again our cells were unsuccessful in taking up the YB3110 plasmid. We concluded that our batch of competent cells were not competent and so these will not be used in any further experiments. A new batch of competent cells are to be generated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

Mammalian Lab

22nd July - MEF passage 6. Training ends.

Week 9

Bacterial Lab

29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.

30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

31st July - A new stock of competent cells was generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.

Ampicillin [link to Ampicillin protocol] was produced and stored. Following miniprep of pSecTag2A [link to miniprep anachem protocol] a gel [link to gel protocol] was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A]. 50X TAE diluted to 1X [link to dilution protocol].

Item	Volume (ul)
DNA pSecTag2A	5
HindIII	1
Buffer	1
BSA	0.5
dH20	2.5
Total	10

Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.

August

1st August - Results from newly generated competent cells transformed with pSecTag2A:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	10

This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the transformation procedure from yesterday was repeated to see whether the ampicillin was working -> plates left for incubation 37C o/n.

Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.

2nd August - Plates displayed significant colony growth on Amp plates indicating successful transformation. Negative control however displayed slight colony growth - ampicillin not working effectively, new ampicillin was therefore prepared. End of week inventory was recorded and stocks were topped up.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

Mammalian Lab

29th July - Thawed [internal link to mammalian protocol] and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu

30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend

Week 10

Bacterial Lab

5th August - The new batch of ampicillin was tested via transformation of competent cells with pSecTag2A. Plates were spread and incubated 37C o/n.

6th August - Results from yesterday’s transformation:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

This indicates that there is still an issue with the ampicillin, possibly a problem with the stock powder used. A final test with both old and new ampicillin was carried out and compared without plasmid insertion.

7th August -

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 Old Ampicillin	Yes	Yes	100+
2 New Ampicillin v2	No	Yes	100+
3 Positive Control	Yes	No	15

Results indicated that the ampicillin source may not have been fully functional, therefore a new source of amp powder was located and amp was remade.

8th August - Preparation of 4x Amp plates and 4x no drug plates for storage in the fridge.

9th August - Meeting with Darren Nesbeth, requirements for upcoming weeks: create glycerol stocks of both pSecTag2A and pSB1C3, purify and extract pure DNA for pSecTag2A and pSB1C3.

Mammalian Lab

6th August - Checked HeLa cells. Cells are growing slow, left to grow for a few more days

8th August - HeLa cells are about 30% confluent. Changed media.

Week 11

Bacterial Lab

12th August - Chloramphenicol [link to Chloramphenicol recipe] was produced and stored at -20C. In order to produce glycerol stocks, pSecTag2A and pSB1C3 were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37C o/n.

13th August - Results from the following plates:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
pSecTag2A Cells	Yes	Yes	100+
W3110 Cells Amp	Yes	No	0
pSecTag +ve control	No	Yes	100+
PSB1C3 Cells	Yes	Yes	0
W3110 Cells Chlor	Yes	No	25
PSB1C3 +ve control	No	Yes	0

This indicated that the pSecTag2A cells are acceptable to use for glycerol stock [protocol for generating glycerol stocks] generation and also for plasmid purification. Miniprep was performed on the two incubated Falcon tubes from yesterday.

The results concerning pSB1C3 indicated that the chloramphenicol did not work, and the glycerol stock is dead. Therefore a new glycerol stock was sought after and chloramphenicol was remade. Amp & Cmp were remade and pSB1C3 was located in the 2012 distribution kit. Amp & Cmp were both tested by producing 1x positive and 1x negative plate for each antibiotic streaked with W3110 cells ->left to incubate o/n at 37C. Glycerol stock of pSecTag2A was grown, and a miniprep was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.

14th August - Two analytical digests of pSecTag2A with EcoR1-HF and Dpn1 were carried out.

Item A	Volume A (ul)	Item B	Volume B (ul)
pSecTag2A	5	pSecTag2A	5
EcoR1-HF	1	Dpn1	1
Buffer 4	1	Buffer 4	1
BSA	0.5	BSA	0.5
dH20	2.5	dH20	2.5
Total	10	Total	10

[insert image of gel]

[link to enzyme conditions]

The pSB1C3 glyc stock (from HQ) was from the 2012 iGEM box in MMP -20C

Told to use a ratio of 3:1 inoculum:80% glycerol when making glyc stocks

Darren's instructions:

pSB1C3

Transfer entire glycerol tube contents to 10mL LB ND in a 50mL Falcon & measure OD. Place in 37C shakee overnight. Measure OD next morning. If there is growth in the inoculum use a loop to streak onto cmp and ND plates again.

pSecTag2A

50ul of 2013 pSecTag2A glyc stock to inoculate 10mL LB Amp in a 50mL Falcon. Grow overnight and the next day generate 15x glyc stocks sing 1.5mL eppendorfs -> store in MMP -20C.

15th August - OD results for pSB1C3 LB ND inoculum

Plasmid	OD Before	OD After
PSB1C3 (LB ND)	0.052	0.5

15x eppendorfs of pSecTag2A Amp glycerol stock and 4x eppendorfs of pSB1C3 ND glycerol stock was prepared. pSB1C3 LB ND was streaked onto two plates (1x Cmp, 1xND) ->incubate o/n @37C Remaining 2ml of pSB1C3 LB ND (from falcon) is used to purify and conduct an analytical digest (16th August).

Restriction digest of pSecTag2A:

ul	EcoR1 single digest	Spe1 single digest	Double digest	Uncut
pSecTag2A	5	5	5	5
EcoR1	1	0	1	0
Spe1	0	1	1	0
BSA	0.5	0.5	0.5	0.5
Buffer 4	1	1	1	1
dH20	2.5	2.5	1.5	3.5
Total	10	10	10	10

[insert image of gel]

iRRE+PC+RBS and PC+RBS (containing pSB1C3, taken from 2012 iGEM boxes) were plated onto cmp plates -> incubated o/n @37C

Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml cmp -> incushaker o/n

16th August - Falcons were retrieved:

RESULTS

Inoculum:

Falcon contents (+LB+Amp) ul	Colony growth	Absorbance
IRRE+PC+RBS (15ul)	Yes	0.8
IRRE+PC+RBS (5ul)	Yes	0.7
PC+RBS (15ul)/td>	No	0.06
PC+RBS (5ul)	No	0.08

Plates

Plate contents (+LB+Amp) ul	Colony growth	Absorbance
IRRE+PC+RBS	Yes	100+
IRRE+PC+RBS	No	100+
PC+RBS/td>	Yes	0
PC+RBS	No	20

The PC+RBS plates & falcons were discarded

IRRE+PC+RBS (15ul inoculum) -> underwent miniprep

IRRE+PC+RBS (5ul inoculum) -> 4x glycerols were made: 500ul culture + 166ul 80% glycerol, stored in MMP -20C iGEM 2013 box.

pSB1C3 Gel sd: EcoR1 & Pst1 + dd

Item (ul)	EcoR1	Pst1	Double digest	Uncut
pSB1C3	5	5	5	5
EcoR1	1	0	1	0
Pst1	0	1	1	0
Buffer 3	1	1	1	1
BSA	0.5	0.5	0.5	0.5
dH20	2.5	2.5	1.5	4.5
Total	10	10	10	10

3ul loading dye to each solution

Lanes:

2 - Hyperladder, 4 - EcoRI, 5 - PstI, 6 - dd, 7 - Uncut

Results from gel: 1kb DNA ladder showed on gel. No bands for EcoR1, Pst1, DD and Uncut plasmid were seen - indicating that no DNA was present -> possibly due to problems with the miniprep.

Therefore from the iGEM 2012 plate 5 (distribution kit) well 23.O, DNA containing BBa_j04450 (colonies are clearly red in colour - RFP) in pSB1C3 was located [link to protocol for resuspension of dried DNA]. The dried DNA was resuspended in 10ul dH2O, left to sit for 5 minutes, then transformed into competent cells -> streaked onto plates and left to incubate o/n @30C over the weekend.

Mammalian Lab

12th August - HeLa cells have confluency of about 60%. Passaged the HeLa cells and ‘backup’ HeLa cells. We set up two 6-well plates at 0.25 x 10^6 cells/ml in preparation for Zeocin kill curve. Cell count involved (0.45 cells/ml).

13th August -

Prepared media with various concentrations of zeocin for each of 6 wells.

Concentration of zeocin (µg/ml)	Volume of zeocin (ml)	Volume of DMEM + 10% FBS + 2 mM L-Glu (ml)
0	0	30.0
50	15	30.0
100	30	30.0
250	75	29.9
500	150	29.9
1000	300	29.7

T75 flasks: - 90% confluency T25 flasks - 30% confluency 6-well plates - 30% confluency

14th August - T25 flasks 90% confluency

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Comment	Confluency (%)	Cell Appearance	Comment
0	65	Healthy	Healthy, few swell	65	Few floaters	minimal floaters
50	40	Healthy, few swell	few floaters	65	occasional swell	moderate floaters
100	40	Half/moderate swell	Moderate floaters, minor infection	50	40% swelling	minimal floaters
250	25	most/moderate swell	many floaters	60	moderate/severe swelling	many floaters
500	40	most clumps dead/ swell	many floaters, possibly infection	65	severe swelling all over	large number of floaters
1000	45	very severe death/ swell	many floaters, minor infection	60	severe swelling all over	moderate number of floaters

The two T25 flasks with revived HeLa have confluency of about 90%; T75 flask with ‘backup’ cells 70% confluent. The T25 flasks are discarded.

16th August -

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	90	Healthy	Minimal	95	Healthy	minimal
50	80	minor swell	moderate	90	minor swell	moderate
100	65	Minor swell, minor death	Many	75	Minor swell, minor death		moderate
250	50	severe swelling, death	many	50	severe swelling, death	many
500	60	severe swelling, death		many	65	severe swelling, death	many
1000	40	severe swelling, death		many	50	severe swelling, death	many