Team:UCL/Labbook

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.

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, minimal agar, 1.4% molten agar solution and 0.1M CaCl2/15% glycerol were prepared for the generation of competent cells. Minimal agar plates were poured and streaked 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 + 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 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 of our competent cells with plasmid YB3110 was carried out.

23rd July - No colony growth was observed on Ampicillin plates 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 were generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.

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

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.

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 protocol from yesterday was repeated to see whether the ampicilin 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 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 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 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.

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. 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	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.

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

17th August - Passaged HeLa cells

Week 12

Bacterial Lab

19th August - pSB1C3 streaked plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful transformation. Colonies were expected to be red in colour, however this was not observed.

Plate (competent cell vial)	Colony Count
10	50+
20	50+

A single colony from each plate was then inoculated in LB in order to later prepare glycerol stocks.

iGEM 2012 boxes were searched for pSB1C3. Transformation was carried out and then selective plates were spread, incubated o/n @37C.

Innoculations of the following were carried out in 2mL LB:

· IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker

· RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker

^ 1.5mL miniprep, 0.5mL glycerol stock for following day (20th)

20th August - Plates taken from incubation:

Competent cells transformed with plasmid: Colony growth -> cells have successfully taken up plamid.

Negative control (only competent cells): Colony growth -> Most likely problems with Chloramphenicol.

Will therefore re-make chloramphenicol

Plate (pSB1C3+)	Colony Count
NUC	5
LAC	20
LAC	15
LAC2	10
CURL1	30
Control	50

take from incu-shaker:

IRRE+PC+RBS ( pSB1C3) -> 4x 200ul glycerols -> glycerol box -20C mmp

RFP+PSB1C3 -> 4x 200ul glycerols/a> ->glycerol box -20C mmp

Minipreped all 4 falcons and a gel was run -> no bands were seen indicating no DNA present.

Item (ul)	IrreAcut	IrreAuc	IrreBcut	IrreBuc	RFPcut	RFPuc	Uncut	Uncut
pSB1C3	5	5	5	5	5	5	5	5
EcoR1	1	0	1	0	1	0	1	0
Pst1	1	0	1	0	1	0	1	0
Buffer 3	1	0	1	0	1	0	1	0
BSA	0.5	0	0.5	0	0.5	0	0.5	0
dH20	1.5	5	1.5	5	1.5	5	1.5	5
Total	10	10	10	10	10	10	10	10

21st August - Nanodrop of pDNA

Sample	Concentration (ng/ul)	260/280
Irre A pDNA	-33.0	1.74
Irre b pDNA	123.0	0.94
GFP pDNA	-52.1	1.63
LB pDNA	-28.4	1.34

10x cmp plates were made using Yanika's chloramphenicol

Meeting with Darren:

The following Biobricks ordered were brought by our supervisor to be streaked onto the relevant antibiotic plates and inoculated in antibiotic+2mL LB -> Incubate o/n @ 37C.

Biobrick	Plate (+ND)	Inoculation (+ 2mL LB)
J63008	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP
K105028	1X AMP	2 mL AMP
I712004	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP
K105030	1X AMP	2 mL AMP
J63008/9	1X AMP	2 mL AMP
K105027	1X AMP	2 mL AMP
K812014	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP

A PCR of Zeocin was performed and a gel was subsequently ran. PCR tube:

1 - PCR zec bb F, R, 2ul template

2 - PCR zec bb F, R, 1ul template

3 - PCR zec F, R, 2ul template

4 - PCR zec F, R, 1ul template

5 - PCR negative control zec bb F, R,

6 - PCR negative control zec F, R

PCR was unsuccessful

22nd August -

Results from Biobrick streaking and inoculation:

Biobrick	Positive Control (ND)	Cmp plate	Amp plate	Falcon
J63008	Growth	Growth	No Growth	No Growth
K105028	Growth		Growth	Growth
K105027	Growth		Growth	Growth
K105030	Growth		Growth	Growth
K812014	Growth	Growth		Growth
J63008/9	Growth		Growth	Growth
I712004	No Growth	No Growth	No Growth	No Growth

Glycerol stocks were therefore made from the plates that displayed colony growth.

A second attempt at zeocin PCR was performed using Phusion DNA Polymerase

1 - Zec bb F, R 2ul

2 - Zec bb F, R 1ul

3 - Zec F, R 2ul

4 - Zec F, R 1ul

5 - Negative control zec bb F, R

6 - Negative control zec F, R

A gel was run with 8ul of each of the 6 reactions. PCR was successful.

Restriction digest of the following samples:

A - K105028

B - K105027

C - K105030

D - K812014

E - J63009/8

	Double Digest	Uncut
Sample	5	5
EcoR1	1	0
Pst1	1	0
Buffer 3	1	0
BSA	0.5	0
dH2O	1.5	5
Total	10	10

Nanodrop of samples:

Sample	260/280	ng/ul
K105028	2.12	39.0
K105027	1.99	47.1
K105030	2.07	48.8
K812014	1.96	113.3
J63008/9	1.95	59.7

PCR Purification

1 - zeo bb F, R 2ul template

2 - zeo bb F, R 1ul template

^ stored in iGEM 2013 box

Preparative digest of K812014 (pSB1C3)

Sample	5ug
E	7
P	7
B3	10
BSA	2
dH2O	30
Total	100

5ug sample = 44ul of 113.3 ng/ul

CMV PCR

Primers used: 2s + 6FW, 2s + bbRE

23rd August -

Gel was ran with (lane):

(3) PCR purified zeo bb FR + 2ul template

(4) PCR purified zeo bb FR + 1ul template

(6) bwf + bb RE + 1ul template

(7) bwf + bb RE + 2ul template

(8) bwf + bb RE

Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:

Sample	260/280	ng/ul
PCR purified zeo bb FR + 2ul template	1.66	55.6
PCR purified zeo bb FR + 1ul template	1.82	17.2

Mammalian Lab

19th August - Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	100	Over confluent	Moderate	100	Over confluent	moderate
50	50	Moderate swelling and death	Many	80	Moderate swelling and death	Many
100	20	severe swelling, death	Many	20	severe swelling, death	many
250	0	-	many	0	-	many
500	0	-	many	0	-	many
1000	0	-	many	0	-	many

Split stock HeLa cells

20th August - Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	100	Over confluent and death	Moderate	90	Over confluent	moderate
50	80	Over confluent, swelling and death	Many	20	Moderate swelling and death	Many
100	20	severe swelling and death	Many	0	severe swelling, death	many
250	0	-	many	0	-	many
500	0	-	many	0	-	many
1000	0	-	many	0	-	many

21st August - Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml

22nd August - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml

Week 13

Bacterial Lab

27th August -

Purify zeocin CMV from zeo FB primers

Zeocin and CMV purification

Tube:

1 - CMV 1ul template bb F, R

2 - CMV 2ul template bb F, R

3 - Zeocin 2ul template zecF, R

4 - Zeocin 1ul template zecF, R

5 - Zeocin 2ul template zecbbF, R

6 - Zeocin 1ul template zecbbF, R

28th August - 3x glycerol stocks of K812014 were prepared from inoculum. 1.5mL of culture was used for miniprep.

Nanodrop of Zec and CMV was recorded:

	260/280	ng/ul
ZEC	1.68	21.1
CMV	1.96	82.1

A gel was run on 100ul of prep digest K812014 + 20ul dye. 2000bp was scooped in gel extraction.

Chloramphenicol was tested at x2 and x4 concentration with 50ul cells spread on each plate, results from the next day still showed significant colony growth (100+).

29th August - Nanodrop of pSB1C3 purified from gel extract, 260/280: -25/21, ng/ul: 13.5

5ul was run on a gel, however extract of pSB1C3 was unsuccessful as no bands were visible.

PCR:

3 PCR reactions of zeocin using zec BB F, R primers were performed. First reaction: 1 ul pSecTag2A template, second 2 ul pSecTag2A and third reaction - negative control - no template.

3 PCR reactions for cmv promoter were prepared using the same variation of template volume as above. These were tested the next day and were successful. Gel photo (30/08).

Prep digest+gel+extract+purify+nanodrop+5ul gel of zeocin and CMV were carried out

Prep digest:

Sample (ul)	Zeo	CMV	Cyc100	Cyc100	Cyc70	Cyc70	Cyc28	Cyc28
V. Sample	48	45	17.5	17.5	23	23	23.5	23.5
B3	10	10	5	5	5	5	5	5
E	7	7	5		5		5
P	7	7	5	5	5	5	5	5
X				5		5
BSA	2	2	1	1	1	1	1	1
H2O	26	29	16.5	16.5	11	11	10.5	10.5
Total	100	100	50	50	50	50	50	50

Glycerol stocks (x15) of J63009, K105027, K105028 and K105030 were made.

Additionally, nanodrops were recorded:

Sample	260/280	Absorbance	ng/ul
J63009	2.10	0.774	88.7
K105030	209.00	0.653	60
K105028	2.14	0.408	50
K105029	2.18	0.585	77.0
K218014	2.12	0.946	104.9

A prep digest was conducted on K812014 to extract pSB1C3.

Chloramphenicol was tested again but this time with x8 cmp (80ul cmp to 10mL LB agar). Overgrad, undergrad and Yanika chloramphenicol stocks were used and 50ul competent cells were spread -> incubate o/n @37C.

Zec and CMV nanodrops were recorded:

Sample	260/280	Absorbance	ng/ul
ZEC	1.87	9.538	2.5
CMV	2.06	11.048	25.4

30th August

Chloramphenicol test - plate colony growth observation and count: all 3 plates prepared using the chloramphenicol from postgrads, us and Yanika had 30+ very small colonies.

After overnight incubation, from each of the the 10 ml inoculations of K812014 (I), pSecTag2A and second (different) sample of K812014 (II), 2.5 ml were minipreped while the rest of 7.5 ml from each was used to make glycerol stocks.

Nanodrop of these minipreps:

Sample	ng/ul	260/280	260/230	Absorbance
K812014 (I)	117.8	2.10	2.49	0.944
pSecTag2A	35.9	1.81	1.44	0.496
K812014 (II)	10.7	1.71	2.55	0.084

Prep digest of K812014 (I) - miniprep sample prepared by Andy. Contents of reaction:

Sample K812014 (I)	30 ul
EcoRI (E)	5 ul
PstI (P)	5 ul
BSA	1 ul
Buffer 3	5 ul
Total	4 ul
Total Volume of Reaction	50 ul

A gel was run with the E, P cut K812014 (Gel photo?) and the corresponding band of liniarised pSB1C3 (backbone of K812014) was extracted for purification.

Nanodrop of the purified pSB1C3 DNA showed a concentration of 8.4 ng/ul (260/290 = 2.91; 260/230 = 0.02, Absorbance = 7.379) while the nanodrop of the pSB1C3 DNA offered by the postgrads showed 13.01 ng/ul (260/280=1.34).

PCR:

8 new reactions were prepared for zeocin - same primers bb F,R as used previously.

A checking (analytical) gel was performed revealing no bands for reaction tubes 6 and 7 (each of these with 1 ul template)- PCR unsuccessful.

These amplified zeocin together with the 2 reaction tubes of amplified zeocin f aacmv from a day before and a were loaded onto the gel and the bands corresponding to 2 kb were extracted for purification.

31 August -

Gel extraction purification of zeocin and cmv. We are expecting that through this procedure the Phusion polymerase, DMSO, buffer, template DNA will be washed away, thus achieving purification of zeocin and cmv DNA (which are to be digested hence, prepared for ligation in the pSB1C3 backbone).

PCR:

Zeocin was amplified again, in 9 tubes (same reaction) -> a total volume of 50 ul x 9 = 450 ul.

Prep digest of zeocin and cmv straight after PCR.

Components	Prep digest Cmv	Prep digest Zeocin
DNA sample	60 ul	60 ul
EcoRI	7 ul	7 ul
PstI	7 ul	7 ul
Buffer 3	10 ul	10 ul
BSA	2 ul	2 ul
Water	14 ul	14 ul
Total	100 ul	100 ul

These digests were used for LIGATION 1

LIGATION 1: using

pSB1C3 digested and purified	10 ng/ul
Zeocin digested and purified	55 ng/ul
Cmv digested and purified	20 ng/ul

Ligation of zeocin to pSB1C3:

Zeocin (55 ng/ul)	3 to 1	6 to 1
Water (ul)	2.1	0
Quick ligase buffer (ul)	10	10
Backbone (ul)	5	5
Insert (ul)	2.7	5.5
Ligase (ul)	1 ul	1 ul
Total (ul)	21	21.5

Ligation of cmv to pSB1C3:

(20 ng/ul)	3 to 1	6 to 1
Water (ul)	2.4	0
Quick ligase buffer (ul)	10	10
Backbone (ul)	5	5
Insert (ul)	2.6	5.3
Ligase (ul)	1 ul	1 ul
Total (ul)	21	21.3

Controls used:

Controls	1 check no circular backbone	2 check no digestion process	3 control (uncut backbone)
Water (ul)	5	6	9
Quick ligase buffer (ul)	10	10	10
Backbone (ul)	5	5	2 of uncut backbone
Insert (ul)	0	0	0
Ligase (ul)	1	0	0
Total (ul)	21	21	21

5 ul of each of each ligation reaction were used to transform our home-made competent cells (4 vials - all ligations apart from the 3 controls) as well as Yanika’s top 10 cells (7 vials - all ligations).

T10 - Top 10 cells

HM - home made competent cells

	T10	T10	T10	T10	T10	T10	T10	HM	HM	HM	HM
Cell vial no.	1	2	3	4	5	6	7	28	45	51	52
Vol. ligation plated (ul)	90 & 10	90 & 10	100	100	100	90 & 10	90 & 10	100	90 & 10	100	100
Ligation containing	zeo 3:1	zeo 6:1	control 1	control 2	control 3	zeo 3:1	zeo 6:1	zeo 3:1	zeo 6:1	zeo 3:1	zeo 6:1
Cell counts	0	0	0	0	0	0	0	15	0 & 10*	15	15

* 0 colonies in plate with 10 ul inoculum spread and 10 colonies for the 90 ul inoculum spread.

After the transformation protocol we spread the transformed cells on 4x cmp (?) agar plates using both 90 ul and 10 ul cell inoculum in the case of cell vials 1,2,6,7 (Yanika’s) and 45 (ours).

We picked 5 colonies from each plated cell vials 28, 45, 51 and 52 (2 ml and 2 ul cmp) and incubated for 10 hours. These inoculations were then used to make glycerol stocks and minipreps.

Gel extraction purification

The 9 tubes of amplified zeocin performed were loaded on a gel and the 2 kb bands were subsequently extracted (6.2 g of gel). This zeocin DNA was purified using gel extraction purification kit and 3 tubes of each 60 ul purified zeocin resulted (labelling zeo g.e.p.).

Miniprep of K812014 cell-containing-biobrick: in order to make stocks of pSB1C3, the backbone into which this biobrick is inserted.

Nanodrop of purified zeocin (from above):

Zeo g.e.p. tube	ng/ul	260/280
1	36.1	1.95
2	38.9	1.90
3	42.9	1.90

Nanodrop of miniprep of K812014: 197 ng/ul (260/280 = 1.94) Mammalian Lab

31 August - HeLa cells confluency: 100% and 40%. Split 100% confluency dish in 1:4, 40% confluency dish in 1:2

Week 14

Bacterial Lab

1st September

The 20 inoculations were retrieved from the incu-shaker. All falcons showed growth apart from one inoculation from cell vial 28 (ligation 3:1 zeo).

1.5 ml of each tube was used for miniprep while the other 0.5 ml was used to make glycerol stocks.

Prep digest of K812014 (miniprep prepared on the 31 of August)

Component	Volume added (ul)
K812014 DNA	25
EcoRI	5
PstI	5
Buffer 3	5
BSA	1
dH2O	9
Total	50

Nanodrop result of the miniprep of transformations:

Cell vial/colony no.	ng/ul	260/280
28/1	84.2	1.65
28/2	58.5	1.77
28/3	49.6	1.83
28/4	14.6	1.97
45/1	130.9	1.72
45/2	18.7	2.34
45/3	49.6	1.73
45/4	111.3	1.64
45/5	23.9	1.96
51/1	69.5	1.70
51/2	60.9	1.78
51/3	41.2	1.85
51/4	50.9	1.79
51/5	23.9	1.96
52/1	40.4	1.83
52/2	40.2	1.87
52/3	78.1	1.94
52/4	127.2	1.62
52/5	16.5	2.11

Analytical digest of the above minipreps - incubated at 37 degrees C for 1 hour

Volume (ul)	Single Digest (PstI)	Double Digest (EcoRI + PstI)
Miniprep	5	5
EcoRI	0	1
PstI	1	1
Buffer 3	1	1
BSA	0.5	0.5
dH2O	2.5	1.5
Total	10	10

These digests were loaded into a gel in the following order: single digest, double digest, uncut. The result of this gel was negative, there were no bands for the loaded DNAs (the 1 kb DNA ladder was on the gel), the procedures of achieving recombinant zeocin and cmv plasmid were unsuccessful.

We suspect that the water was contaminated with nuclease.

2nd September

Bacterial Lab

We re-autoclaved water due to our suspicion it was nuclease contaminated.

New inoculations (in 3 ml LB) of the transformations were prepared: 5 using cmv ligation and 5 using the zeocin ligation. These were left over night into the incushaker.

The falcons were removed from the incu-shaker. Glycerol stocks were prepared using 0.5 ml while the rest was kept for minipreping.

Preparative digest of zeocin and K812014 plasmid

Components (ul)	Prep digest of Zeocin	Prep digest of K812014
Zeo/K812014	80+	20+
EcoRI	10	5
PstI	10	5
Buffer 3	15	5
BSA	3	1
dH2O	22	14
Total	150	50

These were incubated for 1 hour at 37º C and for 20 min heat inactivated.

Nanodrop of the minipreps from the new inoculations

pDNA new	ng/ul	260/280
51/1	9.5	2.2
51/2	9.9	1.91
51/3	8.9	1.93
51/4	8.6	2.31
51/5	13.2	2.49
45/1	8.2	1.89
45/2	8.7	2.65
45/3	10.7	2.13
45/4	9.1	2.00
45/5	9.7	2.34
28 glycerol stock	40.4	2.23
45 glycerol stock	36.7	2.24
51 glycerol stock	45.4	2.18
52 glycerol stock	28.4	1.98

These samples of minipreps and from glycerol stocks were run on a gel in order to check if the circularised potentially recombinant plasmid is present. Again, there were no bands for any of these samples, hence we tend to believe the ligations were not successful OR/AND the transformation was faulty.

Gel extraction of digested E and P - K812014 biobrick plasmid: After gel extraction purification of the corresponding 2 kb band, the concentration showed up to around 6.6 ng/ul and 260/280 = 1.73. Given the very small concentration, a second attempt of obtaining pure pSB1C3 was performed, not successful though, only 4.9 ng/ul concentration.

Gel extraction of PCR purified zeocin, 2 kb band. This was purified and the nanodrop showed a concentation of 77.6 ng/ul and 260/280 = 1.82.

Nanodrop of E, P digested cmv (from 30 of August) gave a concentration of 25.1 ng/ul, 260/280 = 1.86.

New inoculations of K812014 biobrick were prepared and left for incubation over night in the incu-shaker (37 degrees Celsius, at 200 rpm). 4th September Miniprep of the K812014 biobrick inoculations showed the following concentrations:

Mammalian lab

2nd September - All 6 dishes have 100% confluency. Split 3 dishes 3:1 obtaining a total of 9 dishes. Discarded 3 dishes.

pDNA new	ng/ul	260/280
1	151	1.93
2	80.8	1.93
3	111.5	1.82
4	219.1	1.94

Samples 1 and 2 went through a preparative digest.

Component	Volume added (ul)
pDNA	50
EcoRI	7
PstI	7
Buffer 3	10
BSA	2
dH2O	24
Total	100

Ligation 2:

pSB1C3 digested and purified, concentration = 25 ng/ul

Zeo digested and purified conc = 77 ng/ul

CMV digested and purified conc = 25 ng/ul

CMV (25 ng/ul)	1 c (3:1 molar ratio)	2 c (2:1 molar ratio)	3 c (1:1 molar ratio)	4 c (3:1 molar ratio)
Water (ul)	1.8	3.2	8	6
Quick ligase buffer (ul)	10	10	10	10
Backbone (ul)	4	4	1	1
*Insert (ul)	4.2	2.8	1	3
Ligase (ul)	1	1	1	1
Total	21	21	21	21
water (ul)	1.8	3.2	8	6
Colony count	50	100+	100+	150+

Zeocin (77 ng/ul)	5 z (3:1 molar ratio)	6 z (2:1 molar ratio)	7 z (1:1 molar ratio)	8 z (3:1 molar ratio)
Water (ul)	2.1	3.3	8	8
Quick ligase buffer (ul)	10	10	10	10
Backbone (ul)	4	4	1	1
*Insert (ul)	3.9 from *	2.6 from *	1 from *	1
Ligase (ul)	1	1	1	1
Total	21	21	21	21
water (ul)	1.8	3.2	8	6
Colony count	30	0	1	0+

* we prepared a tube of concentration 25 ng/ul

Controls	9	10	11
Water (ul)	6	7	7
Quick ligase buffer (ul)	10	10	10
Backbone (ul)	4	4	4 of uncut
*Insert (ul)	0	0	0
Ligase (ul)	1	0	0
Total	21	21	21
Comments	Check no circular backbone	Check digestion
Colony count	0	0	0+

5 ul of each ligation was used to transform our home made competent cells. After the transformation procedure, these cell vials were entirely spread on 2 x cmp plates.

The 2 ordered oligonucleotides (1gm Oxn LF and 1gm ox LF) were annealed according to the open wet lab protocol. The thermocycler was busy at the time we booked therefore we thermocylced in the evening left in the machine at 4 degrees Celsius over night to collect the following morning.

5th September

The pSB1C3 obtained from the postgrads was digested.

1 colony per each plate of transformation was picked and inoculated in 2 ml LB with 4 ul cmp. These were incu-shaked over night (37 degrees Celsius and 200 rpm).

PCR of zeocin and cmv was performed: 8 same reactions for zeocin, 3 same reactions for cmv and 2 controls (no pSecTag2A template). The zeocin and cmv reaction tubes contained a volume of 1.5 ul template. Primers used for zeocin: zec bb F,R and for cmv: bFW and bbRE.

Ran out of any Phusion DNA polymerase.

OxLF and OxLF Annealing oligos:

The annealed oligos were retrieved from the thermocycler in the morning. The entire tube contents (30 ul) underwent PCR purification - producing aprox. 30 ul pure DNA. 5 ul from this volume was run on a 3% agarose gel with both a 50 bp and 1 kb DNA ladder (120 V for 1 hour). The gel showed the annealed oligos at the right length. The left 24 ul of the pure DNA tube were prep digested while 1 ul was used for nanodroping ( 318.6 ng/ul, 260/280 = 1.84).

Prep digest of purified DNA oligos.

Component	Volume added (ul)
Sample	24
EcoRI	5
PstI	5
Buffer 3	10
BSA	2
dH2O	4
Total	50

These digests were run on a gel and the correct bands were extracted.

Mammalian Lab

All 9 dishes are over-confluent, some with floating dumps. One dish flamentous contamination.

Discarded 5 dishes, including contaminated dish.

Split 4 dishes in 1:4. 16 dishes in total (P15).

6th September

Bacterial Lab

Did glycerol stocks and minipreps of the new transformations.

Nanodrop results of the miniprep:

	1 c	2 c	3 c	4 c	5 z	7 z
ng/ul	7	12.2	17.3	14.1	18.6	13.8
260/280	1.78	1.69	1.87	1.98	1.88	1.68

Analytical digest of the above minipreps:

Component	Volume added (ul)
pDNA	5
EcoRI	1
PstI	1
Buffer 3	1
BSA	0.5
dH2O	1.5
Total	10

These digestions were run next to uncuts but again, no bands on the gel.

Gel extraction and purification of: EcoR1 and Pst1 (E and P) digested K812014 (2 kb band corresponding to pSB1C3) was purified and the nanodrop results were: 51.1 ng/ul and 260/280 = 1.88 OxLF and OxLF annealed. Nanodrop result: 44.6 ng/ul and 0.08 for 260/280. Mammalian Lab

The incubator was decontaminated . Confluency was between 30-90%.

Split 4 dishes in 4:1, 5 dishes in total (P16).

Discarded 1 dish (not enough tryptophan). Other 11 are kept incubated.

7th September-

Picked a total of 15 colonies from plates of potentially ligated cmv and zeocin in pSB1C3 - from both transformations (31st of August and 4th of September). Incubated overnight for 16 hours in 2 ml LB broth and 4 ul chloramphenicol (cmp). Made new glycerols stocks using 0.5 ml of the inoculated volume. Minipreped the 15 inoculations but the nanodrop of these showed concentrations of below 20 ng/ul.

Week 15

9th September -

Bacterial Lab

Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.

Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.

Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.

LIGATION 3:

pSB1C3 digested and purified, concentration = 25 ng/ul

zeo digested and purified conc = 77 ng/ul

CMV digested and purified conc = 25 ng/ul

Zeocin (77 ng/ul)	3 to 1 (mass ratio) [z1]	6 to 1 (mass ratio) [z2]
water (ul)	6	4
Quick ligase buffer (ul)	10	10
backbone (ul)	2	2
insert (ul)	2	4
ligase (ul)	1	1
Total	21	21

Cmv (25 ng/ul)	3 to 1 (mass ratio) [c3]	6 to 1 (mass ratio) [c4]
water (ul)	2	3
Quick ligase buffer (ul)	10	10
backbone (ul)	2	1
insert (ul)	6	6
ligase (ul)	1	1
Total	21	21

Controls	5 ctrl check no circular backbone	6 ctrl check digestion process	7 ctrl (uncut backbone)
water (ul)	7	18	18
Quick ligase buffer (ul)	10	0	0
backbone (ul)	3	3	3 of uncut backbone
insert (ul)	0	0	0
ligase (ul)	1	0	0
Total	21	21	21

These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.

Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).

These were incubated overnight and then minipreped.

10th September -

Bacterial Lab

Colony counts of the re-transformation of ligations:

Plate	Colony counts
2z from 31/08	100
5z from 4/09	150+
6z from 4/09	80+
7z from 4/09	100
8z from 4/09	0
9z - ctrl from 4/09	40
11z - ctrl from 4/09	0

MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock.

Colony count - 90% - 100+ and 10% 80 colonies.

Mammalian Lab

Threw away 12 over-confluent ( aprox. 150%) plates (P15).

5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.

Added FBS and PS intp DMEM media to make new solution.

10th September -

Bacterial Lab

Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone.

There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.

Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.

The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.

The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes. The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.

Building up the competent cell stock using the home-made competent cells

Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread

Plate number	Drug	Volume competent cells (ul)	Colony count
I	1000 X CMP	20	60
II	500 X CMP	20	15
III	250 X CMP	20	0
IV	N/A	20	100+
V	1000 X CMP	20	10
VI	500 X CMP	20	3
VII	250 X CMP	20	0
VIII	N/A	20	100+
IX	1000 X CMP	100 from T vial	15
X	N/A	100 from T vial	100+
I	1000 X CMP	20	60

Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.

New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.

Transformation number 3:

We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.

The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.

After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.

On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.

11th September

Bacterial Lab

8 colonies were inoculated for each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations) The 32 noculations were minipreped and nanodropped. Samples with nanodrop reading above 30 ng/ul were used for analytical digest (16 samples) Colony counts of the transformed cells with the ligation prepared on the 9/09:

Plate	Colony count
Z1	12
Z2	0
C4	1 (also growth of transparent and very small colonies)
5	50
6	50
7	17
pSecTag2A	10 (more transparent than others)

Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).

Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).

Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands:

- For zeocin 2 bands: one of 1037 bp and another of 783 bp

- For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp

Gel showed expected bands

DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul)	5 ul
Buffer 4	1 ul
Water	3 ul
StuI	1 ul
Total	10 ul

The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul).

12th September -

Prep digested samples from minipreped inoculations 1, 14, 16, 17, 24 using StuI

After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.

Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.

Ligations (ul)	AA1	BB1	CC1
EP cut, purified pAZEC fragment (53 ng/ul)	2	0	2
EPD cut pSB1C3*	2	2	0
T4 DNA ligase buffer	1	1	1
Vol. of dilute T4 ligase**	1	1	1
RO water	4	6	6
Total	10	10	10
Cell counts	0	0	0

* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;

** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex.

These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.

Transformation 4:

Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific transformation protocol for this type of cells was followed ([http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf]) apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.

3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.

13th September-

PCR of bb zeocin using Taq PCR kit New England Biolabs in 50 ul reaction volume. (protocol [https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction])

Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09

Sample	Ng/ul	260/280
Z1.1	24.8	1.8
Z1.2	35.4	1.98
Z1.3	34.2	1.93

Spreaded straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.

· Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.

· Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).

· New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.

Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band).

14th September -

Colony counts of the transformation prepared on 12 and 13 of September.

Plate and cmp conc.	AA1 5X CMP	AA1 2X CMP	AA1 1X CMP	BB1 5X CMP	BB1 2X CMP	BB1 1X CMP	CC1 5X CMP	CC1 2X CMP	CC1 1X CMP
Transformation of 12/09, spreading of leftovers	1	0	-	3	28	-	0	0	-
Transformation of 13/09	0	-	30	0	-	3	0	-	0

8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.

Nanodrop result of these minipreps – data from the next day

AA1 1xcmp colony number	Ng/ul	260/280
1	26.8	1.86
2	24.8	1.94
3	35.1	1.9
4	28.2	1.95
5	30.5	1.94
6	28.7	1.99
7	35.1	1.85
8	40.3	1.88

The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.

PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.

A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA. 15th September

Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.

From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.

Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):

Volumes (ul)	MMP-9 4bb	MMP-9 4bb	Control (no backbone)	Control (no insert)	MMP-9 3
Water	2.5	2.5	5	7.5	2.5
pSB1C3	2.5 (HS)	2.5 (sourced from K812014	0	2.5 (HS)	2.5 (HS)
T4 Ligase buffer	10	10	10	10	10
T4 Ligase	1	1	1	1	1
Insert (MMP-9)	5	5	5	0	5
Total	21	21	21	21	21
Vial labels	1	2	3	4	5

Week 16

16th September -

Results of the inoculations of transformations of 13/09 in 4xcmp: all showed growth apart from falcons 5.3, 5.2, 1.14 and 1.2. Glycerol stocks of the rest of 19 inoculations were made.

Minipreps of the above inoculations were made only for 1.3, 1.7, 1.9, 1.10, 1.11, 1.15, 1.20 and 5.1 due to lack of chromatographic columns.

Nanodrop result of the above minipreps showed concentration values below 16.0 ng/ul.

Analytical digest of miniprep samples 1-9 (prepared the day before) as well as of 5.1 and 1.15 (which showed concentrations of about 15 ng/ul) with E and P