iGEM 2015 — Team IISER_Pune

# Team:IISER Pune/Notebook

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---

## List of Note Books

[* Hijack Module (Fast robust oscillator) Notebook](#Hijack_Module)
[* Detection Chromoproteins Notebook](#Chromoproteins)
[* Detection Carotenoids Notebook (pdf)](https://static.igem.org/mediawiki/2015/2/29/IISER_PUNE_Notebook_Carotenoids.pdf)
[* Detection Enzyme Substrate Notebook](#Enzyme_Substrate)
[* Termination notebook(pdf)](https://static.igem.org/mediawiki/2015/9/97/IISER_PUNE_Notebook_Terminator.pdf)

# Hijack Module (Fast robust oscillator) Notebook

25th 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.

26th 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.

27th 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.

28th June 2015

No growth was observed in both the liquid cultures.

A new colony was inoculated again.

29th 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

1st 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.

2nd July 2015

BBa\_K094120 arrived from iGEM Headquarters and was inoculated in 5ml LB media +
5μL Ampicillin. Kept at 37°C for 12 hours.

3rd July 2015

The liquid culture of BBa\_K094120 was plated and kept
overnight at 37°C

5th July 2015

A single colony was inoculated for BBa\_K094120. Kept at 37°C
for 12 hours

6th 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 |

29th 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.

11th 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

12th 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.

17th August 2015

Primary cultures used to make an overnight 5ml culture of
BBa\_C0080, BBa\_C0012 and BBa\_K094120.

18th 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.

21st August 2015

Inoculated BBa\_I13504 (GFP, used as reporter in oscillator
construct) from previously transformed plate. Kept at 37°C for 12 hours

22nd 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

23rd August 2015

Inoculated a colony for BBa\_B0034 and kept at 37°C for 12
hours.

24th August 2015

Plasmid Isolation using Qiagen Miniprep kit:

|  |  |  |
| --- | --- | --- |
| Sample | Concentration (ng/μL) | A260/A280 |
| BBa\_B0034 | 95.2 | 1.95 |

25th August 2015

-
Transformation of the biobrick
BBa\_B0015 (double terminator) in DH5α competent cells (Chloramphenicol
antibiotic)

-
Plates kept at 37°C for 14 hours

26th 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

28th August 2015

Plasmid Isolation of BBa\_B0015 using Qiagen
Miniprep kit:

|  |  |  |
| --- | --- | --- |
| Sample | Concentration (ng/μL) | A260/A280 |
| BBa\_B0034 | 72.3 | 1.95 |

29th 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.

6th 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.

7th September 2015

-
Transformation of the ligation mix into
DH5α cells

-
Kept at 37°C overnight

8th 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

9th 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.

10th 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

11th September 2015

Ligation set for 2 hours and then transformed.

12th 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.

13th 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.

18th September 2015

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

[[top](#top)]

# 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 |

![](Chromoproteinsw_files/image002.gif)

|  |  |
| --- | --- |
|  |  |
|  |  |

**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 |

![](Chromoproteinsw_files/image004.gif)

![](Chromoproteinsw_files/image006.gif)

**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 |

[<top>]

# Detection Enzyme Substrate Notebook

DETECTION: Enzyme substrate reaction

Parts:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | Biobrick | Part | Size(bp) | Location |
| 1 | BBa\_K1509003 | Constitutive promoter | 2129 | Ordered from iGEM HQ |
| 2 | BBa\_K823016 | RBS + full length LacZ | 5163 | Kit plate 2 |

5/08/15

Transformation of BBa\_K823016 (LacZ gene along RBS) plasmid in DH5α competent cells

Protocol: iGEM Transformation protocol

Master plate is made

6/08/15

Observed colonies on the plate

12/08/15

Liquid culture is prepared for BBa\_K823016

Protocol

* 5ml of LB in 15ml falcon
* Add 5µl of cam
* Pick a colony from the plate and dip in falcon
* Incubate for 12-14hrs

22/08/15

BBa\_K1509003 (promoter) is ordered and got from iGEM HQ. NEB10 is the cell line

Preparation of liquid culture

* 5ml LB in 15ml falcon
* Add 5µl of cam
* A loop of nichrome dipped in NEB10 cells containing promoter plasmid
* Incubate for 12-14hrs

23/08/15

Streaking of plate

* Prepare a cam media plate of 20ml LA
* Streak the plate using previously prepared liquid culture
* Overnight Incubation

24/08/15

Grow liquid stalk culture

* 5ml LB in 15ml falcon
* Add 5µl of cam
* Pick a colony from master plate and dip in media
* Overnight incubation

Preparation of glycerol stalk

* 0.5ml 80% glycerol in cryotube
* 0.5ml bacterial culture
* Flash freeze it using liquid nitrogen

24/08/15

Plasmid isolation by alkaline lysis

Chemicals required:

1. CAM antibiotic
2. LB media
3. Alkaline lysis buffer 1
4. Alkaline lysis buffer 2
5. Alkaline lysis buffer 3
6. Ethanol
7. Phenol: Chloroform (1:1)
8. STE
9. TE with RNase A

Protocol

1. Inoculate 5ml of rich media containing the appropriate antibiotic (CAM)
2. Incubate culture overnight at 37 deg
3. Pour 5ml of culture into a microfuge tube
4. Centrifuge in microfuge for 3 min at 4deg
5. Remove media by aspiration leaving bacterial pellet as dry as possible
6. Resuspend the pellet in 100microl of ice-cold alkaline lysis solution 1 by vortexing
7. Add 200microl of alkaline lysis solution 2 , mix with inverting
8. Add 150 microl ice-cold alkaline lysis solution 3 mix with invertexing
9. Store tube on ice for 3-5 min
10. Centrifuge bacterial lysate ,5 min 13000rpm at 4deg in microfuge
11. Transfer supernatant to fresh tube
12. Add equal volume of phenol: chloroform
13. Mix the organic and aqueous phase by vortexing
14. Centrifuge at maximum speed 2 min at 4deg
15. Transfer the aqueous upper layer to fresh tube
16. Add 2 volume of ethanol
17. Mix by vortexing for 2 min
18. Centrifuge for 5min at 4deg
19. Remove supernatant by aspiration
20. Remove all liquid keeping inverted position
21. Add 1ml of ethanol to pellet
22. Centrifuge for 2min at 4deg
23. Remove all supernatant by aspiration
24. Remove ethanol beads and evaporate it at room temperature
25. Dissolve nucleic acid in 50microl of TE containing RNases A
26. Vortex the solution gently for 2 sec
27. Store DNA solution at -20deg

Result: no DNA pellet after precipitation by ethanol

Comment: Most probably step 12 is done in incorrect way

Inoculation of a colony of BBa\_K823016 and BBa\_K1509003

25/08/15

Plasmid isolation of BBa\_K1509003 using QIAGEN miniprep kit

Protocol: QIAGEN Miniprep gravity column is used

Result: No DNA after precipitation by isopropanol addition

26/08/15

Inoculation of BBa\_K823016 and BBa\_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Results:

|  |  |
| --- | --- |
| part | Concentration(ng/µl) |
| BBa\_K823016 | 52.2 |
| BBa\_K1509003 | 38.7 |

Inoculation of BBa\_K823016 and BBa\_K1509003 for alkaline lysis

27/08/15

Plasmid isolation using Alkaline lysis Miniprep in AB Lab

Chemicals Required: Alkaline lysis buffer 1, 2, 3 buffer 2 freshly prepared, RNases

Protocol: standard protocol is used from sambrook molecular cloning book as mentioned earlier

Result

|  |  |
| --- | --- |
| part | Concentration(ng/µl) |
| BBa\_K823016 | 2650.8 |
| BBa\_K1509003 | 4080.3 |

This has high RNA contamination

Gel Electrophoresis is done which shows high contamination of RNA

2/09/15

Inoculation of both BBa\_K823016 and BBa\_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

|  |  |
| --- | --- |
| part | Concentration(ng/µl) |
| BBa\_K823016 | 275.2 |
| BBa\_K1509003 | 87.3 |

Gel Electrophoresis is done to check isolated plasmid

8/09/15

Inoculation of BBa\_K1509003 in 5ml LB and 5µl cam

9/09/15

Gel Electrophoresis (1%)

Chemicals required: 0.5gm agarose, 50ml TAE, 5µl EtBr

Inoculation of both BBa\_K823016 and BBa\_K1509003 in 5ml LB each

10/09/15

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

|  |  |
| --- | --- |
| part | Concentration(ng/µl) |
| BBa\_K823016 | 735 |
| BBa\_K1509003 | 234 |

Single cut digestion

BBa\_K823016: 1µl

BBa\_K1509003:3µl

|  |  |  |
| --- | --- | --- |
|  | BBa\_K823016 (µl) | BBa\_K1509003 (µl) |
| DNA | 1 | 3 |
| ECoR1 buffer | 1 | 1 |
| ECoR1 enzyme | 0.5 | 0.5 |
| dH2O | 7.5 | 5.5 |
| Total | 10 | 10 |

Gel Electrophoresis: Fig1![](images/image00.png)

12/09/15

 Fig2

Confirmation of plasmid isolated

12/09/15

Digestion for 3A assembly

pSB1K3 which is a Kan backbone  used for 3A assembly

|  |  |  |  |
| --- | --- | --- | --- |
|  |  | BBa\_K823016 (µl) | BBa\_K1509003 (µl) |
| 1 | NEB buffer 2 | 5 | 5 |
| 2 | BSA | 0.5 | 0.5 |
| 3 | EcoR1-HF | 0.5 | 0.5 |
| 4 | Spel | 0.5 | 0.5 |
| 5 | dH20 | 18.5 | 18.5 |
| 6 | Total | 20 | 20 |

Protocol: standard iGEM 3A assembly protocol is used

13/09/15

Ligation using 3A assembly of biobricks

Protocol: standard iGEM 3A assembly protocol is used

15/09/15

Transformation of ligated parts

Protocol: IGEM transformation protocol is used

Result: No colony is observed on both KAN media plates

Comment: most probably failure of digestion

16/09/15

Inoculation of both BBa\_K823016 and BBa\_K1509003

Plasmid isolation using QIAGEN Miniprep spin column

Protocol: QIAGEN Miniprep spin column protocol is used

Result:

|  |  |
| --- | --- |
| part | Concentration(ng/µl) |
| BBa\_K823016 | 102.3 |
| BBa\_K1509003 | 65.5 |

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