Team:UCL/Labbook/Week11

Bacterial Lab

Monday 12th August - Chloramphenicol 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.

Tuesday 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 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 stocks of pSecTag2A was grown, and a miniprep was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.

Wednesday 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.

Thursday 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

Friday 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. 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

Wednesday 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	Healthy	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.

Friday 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	50	severe swelling, death	many
1000	40	severe swelling, death	many	50	severe swelling, death	many

Split and passaged stock HeLa into 2 flasks

Saturday 17th August - Passaged HeLa cells