Hijack Module (Fast robust oscillator) Notebook

25^th June 2015

DH5α competent cells were transformed with the following biobricks:

BioBrick	Part	Backbone	Kit plate, Well
BBa_C0080	araC protein coding sequence	pSB1C3	3,11B
BBa_C0012	LacI protein coding sequence	pSB1C3	pSB1C3

 

The plates were kept at 37°C for 14 hours.

 

26^th June 2015

Transformation results:

Biobrick	Number of colonies
BBa_C0080	0
BBa_C0012	3

 

As number of colonies obtained was very low, the transformation of these biobricks was repeated.

 

27^th June 2015

Transformation results:

Biobrick	Number of colonies
BBa_C0080	3
BBa_C0012	60

 

Inoculation:

A colony from BBa_C0080 plate was inoculated in 7ml LB media + 7μL Chloramphenicol. Same was done for BBa_C0012. These cultures were kept at 37°C for 12 hours.

28^th June 2015

No growth was observed in both the liquid cultures.

A new colony was inoculated again.

29^th June 2015

Growth observed in the cultures of BBa_C0080 and BBa_C0012. Gylcerol stocks were made for both.

Plasmid isolation was done using Alkaline Lysis protocol for the 2 cultures. (1.5ml culture x 3)

Concentrations obtained from nanodrop:

Sample	Concentration (ng/μL)	A260/A280
BBa_C0080 (1)	3301.8	2.02
BBa_C0080 (2)	3670.9	1.98
BBa_C0080 (3)	2777.6	1.95
BBa_C0012 (1)	4560.0	1.97
BBa_C0012 (2)	4542.1	1.94
BBa_C0012 (3)	5123.5	1.61

 

A single digest was set for one of each of these plasmids:

(all volumes in μL)	BBa_C0012	BBa_C0080
PstI	0.3	0.3
10x NEBuffer 3	1	1
100x BSA	0.1	0.1
DNA	0.5	0.5
MilliQ H20	8.1	8.1
Total Volume	10	10

 

-          Kept at 37°C for 2 hours

-          Heat Inactivation: 80°C for 20 minutes

 

Gel Electrophoresis:

-          Digested samples run on a 1% gel.

-          Voltage: 100V

-          Time run: 50 minutes

 

Both samples showed an RNA smear. This occurred as the Alkaline Lysis solution I did not have RNAse. After observing this smear, RNAse was added to the solution.

Re-inoculation of the two cultures was done

 

 

1^st July 2015

A plasmid isolation using Alkaline lysis was done.

Sample	Concentration (ng/μL)	A260/A280
BBa_C0080 (1)	4548.1	1.98
BBa_C0080 (2)	3411.9	2.01
BBa_C0080 (3)	3492.6	1.98
BBa_C0012 (1)	2258.4	1.99
BBa_C0012 (2)	2442.6	2.00
BBa_C0012 (3)	2691.4	1.97

 

A digest was set up with a sample of each plasmid and then run on a gel.

The gel showed bands at correct length, but the band intensity was extremely low and did not reflect the concentration obtained on nanodrop. A new method for plasmid isolation was needed.

2^nd July 2015

BBa_K094120 arrived from iGEM Headquarters and was inoculated in 5ml LB media + 5μL Ampicillin. Kept at 37°C for 12 hours.

 

3^rd July 2015

The liquid culture of BBa_K094120 was plated and kept overnight at 37°C

5^th July 2015

A single colony was inoculated for BBa_K094120. Kept at 37°C for 12 hours

6^th July 2015

Plasmid isolation of BBa_K094120 using Alkaline Lysis:

Sample	Concentration (ng/μL)	A260/A280
BBa_K094120 (1)	1050.8	1.88
BBa_K094120(2)	1493.7	1.95
BBa_K094120 (3)	1365.2	1.97

 

29^th July 2015

1% gel run with all samples for the 3 plasmids BBa_C0080, BBa_C0012, BBa_K094120.

All bands seen had low intensity which did not correlate to the concentration on nanodrop.

 

11^th August 2015

Revive glycerol stocks for BBa_C0080, BBa_K094120 and BBa_C0012. A 50 ml culture was inoculated for BBa_C0080 and BBa_C0012 for a Midiprep using Qiagen kit.

Kept at 37°C overnight

 

12^th August 2015

Midiprep using Qiagen Kit results for BBa_C0080 and BBa_C0012:

Sample	Concentration (ng/μL)	A260/A280
BBa_C0012	7.0	3.62
BBa_C0080	27.8	2.07

 

Since results obtained were very low, a new “Qiagen spin miniprep” kit was obtained.

 

17^th August 2015

Primary cultures used to make an overnight 5ml culture of BBa_C0080, BBa_C0012 and BBa_K094120.

18^th August 2015

Qiagen Spin Miniprep protocol followed. Results:

Sample	Concentration (ng/μL)	A260/A280
BBa_K094120	250.7	1.91
BBa_C0080	165.5	1.92
BBa_C0012	195.4	1.92

 

A digestion was set to check plasmid length:

(all volumes in μL)	BBa_C0080	BBa_C0012	BBa_K094120
DNA	2	2	2
EcoRI	0.2	0.2	0.2
10x EcoRI Buffer	1	1	1
100x BSA	0.1	0.1	0.1
MilliQ H20	6.4	6.4	6.4
Total Volume	10	10	10

 

Gel: 1% gel run at 100V for 40 minutes.

The bands obtained for all 3 plasmids were at the correct length. The uncut plasmid ran faster than cut plasmid.

21^st August 2015

Inoculated BBa_I13504 (GFP, used as reporter in oscillator construct) from previously transformed plate. Kept at 37°C for 12 hours

22^nd August 2015

-          Miniprep of BBa_I13504:

Sample	Concentration (ng/μL)	A260/A280
BBa_I13504	122.4	1.94

 

-          Transformation of the biobrick BBa_B0034 (RBS) in DH5α competent cells (Ampicillin antibiotic)

-          Plates kept at 37°C for 14 hours

 

23^rd August 2015

Inoculated a colony for BBa_B0034 and kept at 37°C for 12 hours.

24^th August 2015

Plasmid Isolation using Qiagen Miniprep kit:

Sample	Concentration (ng/μL)	A260/A280
BBa_B0034	95.2	1.95

 

25^th August 2015

-          Transformation of the biobrick BBa_B0015 (double terminator) in DH5α competent cells (Chloramphenicol antibiotic)

-          Plates kept at 37°C for 14 hours

26^th August 2015

Transformation Result:

Number of colonies for BBa_B0015 = 8

-          Inoculated BBa_B0015 in 5ml LB media + 5 μL Chloramphenicol

-          Kept at 37°C for 12 hours

28^th August 2015

Plasmid Isolation of BBa_B0015 using Qiagen Miniprep kit:

Sample	Concentration (ng/μL)	A260/A280
BBa_B0034	72.3	1.95

 

29^th August 2015

Single Digest to check plasmid length:

(all volumes in μL)	BBa_B0015	BBa_I13504	BBa_B0034
DNA	4	2	2
EcoRI	0.2	0.2	0.2
10x EcoRI Buffer	1	1	1
100x BSA	0.1	0.1	0.1
MilliQ H20	5.7	5.7	5.7
Total Volume	10	10	10

 

Gel: 1% gel run with uncut and cut samples at 100V for 40 minutes.

The 3 single cut bands were at the right length.

 

6^th September 2015

Digestion for 3A assembly of constructs of the oscillator construct:

(all volumes given are in μL)	BBa_ K094120	BBa_ C0080	BBa_ C0012	BBa_ I13504	BBa_ B0034	BBa_ B0015	Ampicillin Backbone	Chloramphenicol Backbone	Kanamycin Backbone
DNA	3	3	3	3	4	5	10	10	10
10x NEBuffer 2	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5	2.5
BSA	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
EcoRI	0.5	0.5	0.5	-	-	-	0.5	0.5	0.5
XbaI	-	-	-	0.5	0.5	0.5	-	-	-
SpeI	0.5	0.5	0.5	-	-	-	-	-	-
PstI	-	-	-	0.5	0.5	0.5	0.5	0.5	0.5
H20	13	13	13	13	12	11	6	6	6
Total	20	20	20	20	20	20	20	20	20

-          Kept at 37°C for 5 hours

-          Heat Inactivation at 80°C for 20 minutes

-          The following devices were to be assembled:

-          BBa_K094120 + BBa_B0034 + Chloramphenicol backbone = Device 2a

-          BBa_C0080 + BBa_B0015 + Kanamycin backbone = Device 2b

-          BBa_C0012 + + BBa_B0015 + Ampicillin backbone = Device 2c

-          BBa_K094120 + BBa_I13504 + Kanamycin backbone = Device 2d

 

 

 

Ligation:

(all volumes in μL)	Device 2a	Device 2b	Device 2c	Device 2d
Ampicillin Backbone	-	-	2	-
Chloramphenicol Backbone	2	-	-	-
Kanamycin Backbone	-	2	-	2
BBa_K094120	2	-	-	2
BBa_B0034	2	-	-	-
BBa_C0080	-	2	-	-
BBa_B0015	-	2	2	-
BBa_C0012	-	-	2	-
BBa_I13504	-	-	-	2
T4 DNA ligase	0.5	0.5	0.5	0.5
Ligase Buffer	1	1	1	1
MilliQ H20	2.5	2.5	2.5	2.5
Total Volume	20	20	20	20

 

Ligation kept overnight at room temperature.

 

7^th September 2015

-          Transformation of the ligation mix into DH5α cells

-          Kept at 37°C overnight

 

8^th September 2015

Transformation results:

Ampicillin Negative Control : Lawn growth

Kanamycin Negative Control : Zero colonies

 

Plate	Number of Colonies
2a	10
2b	12
2c	Lawn growth
2d	5

 

Ampicillin degraded. 2c re-transformed

Inoculation of a single colony from each plate in 5ml LB media + 5μL antibiotic

Kept at 37°C for 12 hours

9^th September 2015

Miniprep by Qiagen Kit:

Sample	Concentration (ng/μL)	A260/A280
2a	283.4	1.91
2b	122.3	2.09
2d	226.6	1.96

 

Single Digest with EcoRI to check plasmid length:

Backbone-Backbone ligation was observed on the gel.

A new ligation was setup. This time insert:vector ratio was increased. Kept at 37°C for 2 hours.

These ligation mix were transformed into Dh5α competent cells.

 

10^th September 2015

Growth observed in negative control plates.

An overnight digestion was set for the individual plasmids again. Kept at 37°C for 12 hours

 

11^th September 2015

Ligation set for 2 hours and then transformed.

12^th September 2015

Colonies of device 2a, 2b and 2c obtained. No colonies on 2d.

3 colonies from each plate were inoculated, incubated for 12 hours at 37°C and miniprepped.

These were then digested with EcoRI to check the length of the plasmid on a gel.

The bands were as expected.

An overnight digest followed by 2 hour ligation was set to assemble to following device:

Device	Part1	Part2	Backbone
5a	2a	2b	pSB1A3
5b	2a	2c	pSB1K3

 

Two samples of 5b were assembled – one low copy and second high copy backbone

BBa_I13504 in Ampicillin backbone was chosen to assemble device 2d. This biobrick was digested and ligated.

Devices 5a, 5b and 2d were transformed.

13^th Spetember 2015

Transformation Results:
                Negative Control: Zero

Plate	Number of Colonies
5a	15
5b (low copy)	70
5b (high copy)	26
2d	10

 

Three colonies were inoculated from each plate, incubated at 37°C and then miniprepped.

These were then single digested using EcorI to check on gel.

2d, 5a and 5b high copy showed correct bands.

 

18^th September 2015

2d construct was characterized by induction with different concentrations of IPTG and arabinose.

Chromoproteins

8th June, 2015

 

●     The following biobrick parts were revived from the iGEM 2015 distribution plates using the iGEM protocol Standard protocol.

●     Transformation was carried with Ultra-competent cells for DH5α strain of E.coli (with 200µL of LB).

●     200µL of the revived product was plated on LA+antibiotic.

●     The plates were kept at 37℃ overnight (12-16 hours).

 

Part Number	Location in Kit	Backbone
J23110 (Constitutive Promoter)	Plate 4, Well 19D	Ampicillin
K592025 (RBS+amilCP)	Plate 1, Well 19C	Chloramphenicol
J04450 (RFP)	Plate 4, Well 2H	Ampicillin

 

 

Negative Control: For both antibiotics (Amp and Cam)

Positive Control: RFP

 

9th June, 2015

 

 

Part Number	Number of colonies
J23110 (Promoter)	Many colonies
K592025 (RBS+amilCP)	Many single colonies
J04450 (RFP)	Lawn growth
Negative Control	No colonies

 

●     Next, the colonies were inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

10th June, 2015

 

●     Growth was observed in K592025, but no growth in J23110.

●     Again a colony was picked from the plate with J23110 and inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

11th June, 2015

 

●     Again no growth was observed in J23110 (even after inoculating twice).

●     Transformation was done again for J23110

●     Different promoter was also transformed.

 

 

Part Number	Location in Kit	Backbone
J23119 (Constitutive Promoter)	Plate 3, Well 17O	Chloramphenicol

 

13th June, 2015

 

●     The two promoters were tranformed with 200µL SOC media which was prepared using the iGEM protocol (earlier transformations with LB).

●     200µL of the revived product was plated on LA+antibiotic.

●     The plates were kept at 37℃ overnight (12-16 hours).

 

14th June, 2015

 

 

Part Number	Number of colonies
J23110 (Promoter 1)	30
K592025 (RBS+amilCP)	3
J23119 (Promoter 2)	47
Negative Control	No colonies

 

 

●     The colonies obtained were inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

18th June, 2015

 

Plasmid isolation was carried out using the Alkaline Lysis Protocol from Sambrook and Maniatis.

Resuspended in TE Buffer.

 

 

 

 

Results of Nanodrop:

 

Sample	Vial Number	A260/A280	Concentration (ng/µL)
K592025	1	1.99	6691.9
	2	2.11	4749.5
	3	2.12	4158.3
J23119	1	2.10	4865.3
	2	2.09	2757.4
	3	2.10	4902.3
J23110	1	2.09	5121.8
	2	2.15	8148.0
	3	2.13	3223.8

 

●     The alkaline lysis buffer I wasn’t added with RNase.

●     The plasmid isolation was repeated with RNase added to Buffer I.

 

 

27th June, 2015

 

●     The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

 

28th June, 2015

 

●     RNase (10mg/mL stock) was added to Alkaline Lysis Buffer I.

●     Resuspension in milliQ water.

 

Results of Nanodrop:

 

Sample	Vial Number	A260/A280	Concentration (ng/µL)
K592025	1	2.13	56.9
	2	1.39	67.4
	3	2.06	58.5
J23119	1	2.16	34.1
	2	2.15	32.3
	3	1.39	116.1
J23110	1	2.08	158.4
	2	2.02	3222.1
	3	1.95	580.4

 

 

29th June, 2015

 

●     The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.

●     They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.

 

30th June-10th July, 2015

Again, alkaline lysis was done.

 

Results of Nanodrop:

 

Date	Sample	Vial Number	A260/A280	Concentration (ng/µL)
30/6	J23119	1	1.94	2376.8
30/6	J23119	2	1.95	2301.2
30/6	J23119	3	1.88	1662.9
8/7	J23119	1	1.99	1616.5
8/7	J23110	2	1.98	618.9
10/7	K592025	1	1.96	1641.1
10/7	K592025	2	1.97	1457.0
10/7	K592025	3	1.99	1404.6

 

 

 

 

 

 

26th August and 3rd September

 

Results for Nanodrop by Qiagen spin mini kit

 

Sample	A260/A280	Concentration (ng/μL)
J23119	2.04	121.1
J23119	1.89	329.3
K592029	1.96	158
K592029	1.88	407.3

 

 

 

 

 

 

26th August

Single digestion reaction

 

(in μL)	K592025	J23119
EcoRI	0.3	0.3
DNA	2	2
10XEcoRI Buffer	1	1
100X BSA	0.1	0.1
Distilled water	6.6	6.6
Total Volume	10	10

 

 

27th August

 

3A assembly double digestion

(in μL)	Promoter(J23119)	amilCP(K592025)	Linear backbone
DNA	2	2	10
10X Buffer2	2.5	2.5	2.5
100X BSA	0.5	0.5	0.5
EcoRI	0.5	-	0.5
SpeI	0.5	-	-
PstI	-	0.5	0.5
XbaI	-	0.5	-
DpnI	-	-	0.5
Distlled water	14	14	5.5
Total Volume	20	20	20

Kept at 37°C for 4 hours,  heat-kill at 80°C for 20min.

Colonies obtained (~10),  were inoculated and miniprep was done with qiagen kit.

 

Table3: Double digestion for 2A assembly

2A assembly Double digestion:

 

(in μL)	Promoter(J23119)	amilCP(K592025)
DNA	7.8	7
10X Buffer2	1	1
100X BSA	0.2	0.2
SpeI	0.5	0.5
PstI	0.5	-
XbaI	-	0.5
Distilled water	0	0.8
Total Volume	10	10