Team:IIT Kharagpur/Notebook

July

Mo Tu We Th Fr Sa Su
29 30 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 31 1 2
3 4 5 6 7 8 9

August

Mo Tu We Th Fr Sa Su
27 28 29 30 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
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

k
DNA(7b) 8µl
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
Department of Biotechnology
IIT Kharagpur

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