iGEM 2015 — Team IIT_Kharagpur

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### July

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
| 29 | 30 | 1 | 2 | 3 | 4 | 5 |
| [6](#6-july) | [7](#7-july) | [8](#8-july) | [9](#9-july) | [10](#10-july) | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | [21](#21-july) | [22](#22-july) | [23](#23-july) | [24](#24-july) | 25 | [26](#26-july) |
| 27 | [28](#28-july) | [29](#29-july) | [30](#30-july) | [31](#31-july) | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |

### August

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
| 27 | 28 | 29 | 30 | 31 | [1](#1-august) | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| 31 | 1 | 2 | 3 | 4 | 5 | 6 |

### September

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
| 31 | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |

## Week One ( July 6 - July 10 )

### 6th July 2015

* LB media prepared (500 ml), autoclaved and stored at 4 degrees.
* 250 ml of LB agar prepared and poured into the plates (chloramphenicol antibiotic used- concentration 1 micro litre/ml)

### 7th July 2015

* DNA from wells dissolved into 10 μl distilled water. DNA kit plate instructions
* Transformed 4μl of DNA into 100μl of E.Coli DH5ɑ. transformation procedure)
* 100μl of transformed cells were plated on chloramphenicol resistant plates.and left overnight to grow.

### 8th July 2015

* Colonies for luxPR promoter (BBa\_R6002) constructs were observed. A colony was picked and inoculated in 3 ml culture for glycerol stock preparation.
* No colony observed for luxR protein (BBa\_K546003) construct.

### 9th July 2015

* Transformation repeated for luxR protein construct .transformation procedure
* Culture prepared for plasmid isolation for luxPR promoter. Colony was picked and inoculated in 10 ml culture.

### 10th July 2015

* No colony observed for luxR protein construct
* Plasmid isolation done for luxPR promoter
* Gel electrophoresis done for luxPR promoter

* Lane 7 : 1kb marker  Lane 8 : luxPR promoter
* Gel electrophoresis done for luxPR promoter

|  | Amount Used | Amount | A 260/280 | A 260/230 |
| --- | --- | --- | --- | --- |
| P1 (luxPR promoter) (BBa\_R6002) | 1 μl | 117.2ng/μl | 1.85 | 1.54 |

## Week Three ( July 21 - July 26 )

### 21st July 2015

* Cells containing LuxR were inoculated in 10 ml culture and kept overnight.

### 22nd July 2015

* LuxR DNA was extracted following the protocol of H1 Media.
* Gel electrophoresis done for LuxR DNA
* Nanodrop done for LuxR.

|  | Amount Used | Amount | A 260/280 | A 260/230 |
| --- | --- | --- | --- | --- |
| P3 (LuxR)  (BBa\_K546003) | 1μl | 191.2ng/μl | 1.91 | 2.14 |

|  | Amount Used | Amount | A 260/280 | A 260/230 |
| --- | --- | --- | --- | --- |
| P3 (LuxR)  (BBa\_K546003) | 1μl | 191.2ng/μl | 1.91 | 2.14 |

* Restriction digestion mix. used for LuxR DNA extracted before. Restriction enzymes used:  1. Xba I  2. Pst I

| Reaction Mixture |
| --- |
| DNA | 10μl |
| Buffer | 2μl (3.1) |
| Pst 1 | 1μl |
| Xba 1 | 1μl |
| H2O | 6μl |
| Kept at 37 degree Centigrade overnight. |

* To check the growth of crt EBI (reporter) in Ampicillin + LB , 3ml culture grown overnight.

### 23rd July 2015

* Glycerol stocks made from 3 ml culture of crt EBI reporter which was grown overnight. (20% glycerol stock kept in 20℃)
* Gel run for restriction digestion of LuxR.
* Inoculation and streaking done for crt EBI reporter.

### 24th July 2015

* Plasmid preparation for crt easeInOutCubic
* Precautions: 1. Use only one column for 10 ml culture while extracting.
  2. After loading the supernatant in the column, wait for 10 min for better DNA binding.
  3. Before eluting with (distilled) autoclaved water wait for 10 min. for better elution
* Nanodrop for crt EBI

|  | Amount Used | Amount | A 260/280 | A 260/230 |
| --- | --- | --- | --- | --- |
| P2 (crt EBI) (BBa\_K274100) | 1μl | 284.2ng/μ | 1.86 | 2.14 |

* Reaction mixture for restriction digestion of crt EBI

|  |  |
| --- | --- |
| DNA | 4μl |
| Buffer | 2μl (3.1) |
| Pst 1 | 1μl |
| Xba 1 | 1μl |
| H2O | 12μl |

### 26th July 2015

* 50 ml culture used for extracting luxPR plasmid using E. coli DH5ɑ strain
* 34 μl/ml of antibiotic (camR) used as a resistance marker
* Plasmid extracted for the LuxPR part from 50 ml culture. 60 μl volume of plasmid obtained.

|  | Amount Used | Amount | A 260/280 | A 260/230 |
| --- | --- | --- | --- | --- |
| P1 (LuxPR) (BBa\_R6002) | 1μl | 170 ng/μl | 1.83 | 2.05 |

* Restriction digestion of LuxPR and crt EBI

|  |  |  |
| --- | --- | --- |
|  | LuxPR | crtEBI |
| DNA | 8μl | 6μl |
| Enzyme 1 | 1 μl (Spe I) | 1μl (Xba I) |
| Enzyme 2 | 1 μl (Pst I) | 1μl (Pst I) |
| Buffer | 2 μl (2.1) | 2μl (3.1) |
| H2O | 8μl | 10μl |

## WEEK Four( July 28 - August 1 )

### 28th July 2015

* Gel was run with crtEBI containing digested plasmid and LuxPR containing digested plasmid  Length of crtEBI ~ 3.3 kbp Length of LuxPR ~ 50 bp  Plasmid containing LuxPR ~ 2.2 kbp
* crtEBI part , and LuxPR containing part extracted from gel . DNA eluted using the kit
* Nanodrop was run

|  |  |  |
| --- | --- | --- |
|  | Amount used | amount |
| LuxPR | 1μl | 28 ng/μl |
| crtEBI | 1μl | 50 ng/μl |

* Ligation performed of crtEBI part into plasmid backbone containing LuxPR.  Insert: crEBI (3.3 kb) , 50 ng/μl  Vector: LuxPR (2.1 kb) , 28 ng/μl

|  |  |
| --- | --- |
| Vector | 6 μl (170 ng) |
| Insert | 2 μl (89 ng) |
| T4 ligase buffer | 2 μl |
| T4 ligase | 1 μl |
| H2O | 9 μl |

### 29th July 2015

* Transformed the ligation product of LuxPR and crtEBI(reporter gene). transformation procedure
* Plating done for the above construct.

### 30th July 2015

* No growth observed for the construct. Steps repeated.
* Restriction digestion of LuxPR: (single digestion)

|  |  |
| --- | --- |
| LuxPR | 7 μl |
| Spe I | 1 μl |
| Buffer (cutsmart) | 2 μl |
| H2O | 10 μl |
| Kept at 37℃ for 5-6 hours. |

* Second digestion setup made.

|  |  |
| --- | --- |
| Product from previous digestion | 20 μl |
| Pst I | 1 μl |
| Buffer (3.1) | 2 μl |
| Kept at 37℃ for 5-6 hours. |

### 31st July 2015

* Gel was run for the restriction digest product.
* Band was cut and DNA eluted using gel extraction kit.
* Ligation set up after purifying the DNA.

### 1st August 2015

* Transformation done for ligated product. Transformation procedure

## Week Five ( August 2 - August 7)

### 2 August

* A single colony was observed.
* The colony was inoculated into a 3 ml Chloramphenicol containing medium.

### 3rd August

* Plasmid extraction done for the colony.
* Screening done by running Gel. The band obtained was below 2 kb size.( Expected band size was 5.4 kb)

### 6th August

* Double digestion performed with Spe I and Pst I.

Reaction Mixture

|  |  |
| --- | --- |
| DNA + Water 20 μl | 20 μl |
| Spe I | 0.5 μl |
| Pst I | 1.0 μl |
| Buffer (2:1) | 2.0 μl |

### 7th August

* Digested Lux PR eluted from gel.
* Plasmid extraction done for Lux PR. Concentration: 126.5 ng/ μl Volume: 55 μl
* Ligation reaction mixture for Lux PR (27 ng/μl) + crtEBI (28 ng/μl)

Reaction Mixture

|  |  |  |
| --- | --- | --- |
|  | V:I = 3:1 | V:I=2:1 |
| crtBI (28.2 ng/μl) | 3μl | 4μl |
| luxPR (27.2 ng/μl) | 6μl | 6μl |
| T DNA ligase | 1.5μl | 1.μl |
| ligase buffer | 1.5μl | 1.5μl |
| Water | 8μl | 7μl |

ligation @ 16 for 16 hours.

|  |  |
| --- | --- |
| crtEBI | 3μl |
| luxPR | 6μl |
| buffer | 1.5μl |
| ligase | 1.2μl |
| water | 3.3μl |

Digestion:
LuxPR (E and S) ­ 3μl CrtEBI (E and X) ­ 1.4 μl

|  |  |
| --- | --- |
| DNA | 12μl |
| EcoRI | 1μl |
| Buffer | 2μl |
| H2O | 5μl |

Sequential

|  |  |
| --- | --- |
| DNA | 10μl |
| EcoRI | 1μl |
| Buffer [Neb buffer EcoRI] | 2μl |
| H2O | 7μl |

Digestion ­ 2

|  |  |
| --- | --- |
| LuxPR | 17μl |
| Spe I | 1μl |
| Buffer (Cutsmart) | 2μl |

|  |  |
| --- | --- |
| CrtEBI | 17μl |
| Xba I | 1μl |
| Buffer (Cutsmart) | 2μl |

Result:
2% gel was prepared since the LuxPR insert was only 55 bp. The gel wasn’t prepared properly.
The same bands were observed twice. This was probably because the concentration was not
uniform throughout the height of the gel. The bands ran faster at the upper surface and slower
at the lower surface.
The 55 bp was not observed at all.

3 A Assembly
Restriction Digestion:
FIRST DIGESTION
Part A

|  |  |
| --- | --- |
| LuxPR (128 ng/μl) | 3μl |
| Buffer (EcoRI Buffer) | 2μl |
| EcoRI | 1μl |
| N.F./ dd H2O | 14μl |

Part B

|  |  |
| --- | --- |
| CrtEBI (140 ng/μl ) | 3μl |
| Buffer (2:1) | 2μl |
| Xba I | 1μl |
| N.F. H2O | 14μl |

A and B kept at 37 for 2 hours
Then PCR Purified.
Backbone

|  |  |
| --- | --- |
| DNA | 5μl |
| EcoRI | 0.5μl |
| Pst I | 0.5μl |
| Dpa I | 0.5μl |
| Buffer (2:1) | 2μl |
| N.F. H2O | 11.5μl |

Kept at 37 for 1 hour
PCR Purification
SECOND DIGESTION

|  |  |
| --- | --- |
| DNA (LuxPR) | 20μl |
| Spe I | 1μl |
| Buffer (Cutsmart) | 2μl |

|  |  |
| --- | --- |
| DNA (CrtEBI) | 20μl |
| Pst I | 1μl |
| Buffer (3:1) | 2μl |

Nanodrop data:
LuxPR ­ 15 ng/μl
CrtEBI ­ 15 ng/μl
Backbone ­ 5 ng/μl
Size of plasmids:
LuxPR ­ 2.1
CrtEBI ­ 5.5
Backbone ­ 2.2
Ligation Mixture:
Molar Ratio­­­ LuxPR : CrtEBI : Backbone = 1 : 1 : 0.5 (Recommended)

|  |  |
| --- | --- |
| LuxPR | 3μl |
| CrtEBI | 8μl |
| Backbone | 5μl |
| Buffer | 2μl |
| Ligase (T4) | 1μl |

Molar Ratio­­­ LuxPR : CrtEBI : Backbone = 3 : 1 : 0.5

|  |  |
| --- | --- |
| LuxPR | 6μl |
| CrtEBI | 5μl |
| Backbone | 3μl |
| Buffer | 2μl |
| Ligase (T4) | 1μl |
| Water (T4) | 3μl |

3A assembly possible plasmids and their sizes for screening
correct plasmid

### 17th August

1-10 b → 1:1:0.5
1-10 a→ 3:1:0.5
7b colony→ plasmid check (68 ng/ μl)
CLONE CONFIRMATION
Digestion 1

|  |  |
| --- | --- |
| DNA (68 ng) | 7μl |
| Buffer (2:1) | 2μl |
| EcoRI | 0.5μl |
| Pst I | 0.5μl |
| H2O | 10μl |

Digestion 2

|  |  |
| --- | --- |
| DNA | 7μl |
| Buffer (CS) | 2μl |
| Xba I | 0.5μl |
| Spe I | 0.5μl |
| H2O | 10μl |

|  |  |
| --- | --- |
| DNA | 7μl |
| Buffer (3:1) | 2μl |
| Pst I | 1μl |
| H2O | 10μl |
| Total | 20μl |

### 3rd September

3A Assembly (Attempt 4)

|  |  |
| --- | --- |
| LuxR | 7μl |
| EcoRI | 1μl |
| Buffer (EcoRI) | 2μl |
| dd H2O | 10μl |

|  |  |
| --- | --- |
| CrtEBI | 10μl |
| Xba I | 1μl |
| Pst I | 2μl |
| dBuffer (3:1) | 10μl |
| dd H2O | 10μl |

Digestion 2

|  |  |
| --- | --- |
| DNA | 7μl |
| Buffer (CS) | 2μl |
| Xba I | 0.5μl |
| Spe I | 0.5μl |
| H2O | 10μl |

|  |  |
| --- | --- |
| DNA | 7μl |
| Buffer (3:1) | 2μl |
| Pst I | 1μl |
| H2O | 10μl |
| Total | 20μl |

### 8th September

|  |  |
| --- | --- |
| DNA | 13ul |
| Spe 1 | 1µl |
| Pst 1 | 1µl |
| Buffer | 2ul |
| dd H2O | 3ul |

### 9th September

|  |  |
| --- | --- |
| DNA(7b) | 8µl |
k| Buffer (EcoR1) | 2µl |
| dd H20 | 9µl |

|  |  |
| --- | --- |
| DNA (CrtEBI) | 10µl |
| Buffer (EcorR1) | 2µl |
| dd H2O | 7µl |

(To get the backbone)

|  |  |
| --- | --- |
| DNA (LuxR) | 10µl |
| Buffer (EcorR1) | 2µl |
| EcorR1 | 1µl |
| dd H2O | 7µl |

#Pst digestion :
#1μl Pst 2μl something
#Kept at 37℃ for 6 hours.

### 10th September

7b : 12 ng/μl
LuxR : 12 ng/μl
LuxPR : 20 ng/μl
CrtEBI : 20 ng/μl

Ligation 1 (standard assembly)

|  |  |
| --- | --- |
| (X/P) CrtEBI (3.3) | 7µl |
| (S/P) LuxPR (2.1) | 5µl |
| T4 Ligase | 1µl |
| Ligase Buffer | 2µl |

Ligation 2 (breakthrough standard assembly)

|  |  |
| --- | --- |
| (X/P) CrtEBI (3.3) | 5µl |
| (S/P) Lux R (3.3) | 8µl |
| T4 Ligase | 1µl |
| Buffer | 2µl |

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Department of Biotechnology
IIT Kharagpur

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