Team:NU Kazakhstan/Project Timeline

Bioremediation of Sour Crude Oil Waste using Cyanobacteria

May

Week 1-4

Week 1	April 30 - May 6
Week 2	May 7 - May 13
Week 3	May 14 - May 20
Week 4	May 21 - May 27

June

Week 5-9

Week 5	May 28 – June 3
Week 6	June 4 – June 10
Week 7	June 11 – June 17
Week 8	June 18 – June 24
Week 9	June 25 – July 1

WEEK 1 (April 30 - May 6)

5 May 2018
BG-11 medium was prepared.

Inoculation of the BG-11 medium with cyanobacteria (Synechococcus elongatus PCC 7942) was done.

WEEK 2 (May 7 - May 13)

11 May 2018
BG-11 agar plates were made.

Cyanobacteria were plated onto BG-11 agar plates from liquid culture

WEEK 3 (May 14 - May 20)

14 May 2018
The new subculture of cyanobacteria was prepared from old liquid culture.

The old liquid culture of cyanobacteria was plated onto BG -11 agar plate.
16 May 2018
BG-11 agar plates were prepared.

Cyanobacteria from liquid culture were plated onto the BG-11 agar plates.

WEEK 4 (May 21 - May 27)

25 May 2018
OD of liquid cyanobacteria culture was measured

Liquid cultures of cyanobacteria were checked.

The new subculture of cyanobacteria was prepared from old liquid culture.

WEEK 5 (May 28 – June 3)

28 May 2018
OD of liquid cyanobacteria culture was measured.

Liquid cultures of cyanobacteria were checked.
30 May 2018
LB liquid medium and LB agar plates were prepared.
31 May 2018
Competent E.coli (TOP10) cells were prepared.

WEEK 6 (June 4 – June 10)

4 June 2018
New competent TOP10 cells were prepared.

Spectinomycin stock solution 1000X was prepared.

LB agar plates with spectinomycin were prepared.
5 June 2018
Transformation of E.coli (TOP10) with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.

Transformed E.coli cells were plated on LB agar plates with spectinomycin.
6 June 2018
LB liquid medium with spectinomycin was prepared.

Inoculation of the LB liquid medium with spectinomycin resistant TOP10 cells was done.
7 June 2018
Plasmids from the transformed TOP10 were extracted by MiraPrep.

Extracted plasmid samples were measured by Nanodrop.

Samples 260/280 260/230 Concentration

1 1.76 1.53 63.96 ng/ul

2 1.96 2.29 1953 ng/u

Gel Electrophoresis was done.
8 June 2018
OD of liquid cyanobacteria culture was measured. OD: 0.8.

BG-11 agar plates with spectinomycin were prepared.

WEEK 7 (June 11 – June 17)

11 June 2018
Inoculation of the LB liquid medium with spectinomycin resistant TOP10 cells was done.
12 June 2018
MiraPrep was done.

Extracted plasmid samples were measured by Nanodrop.

Samples 260/280 260/230 Concentration

1 1.90 2.38 1600 ng/ul

Gel Electrophoresis was done.

Inoculation of the LB liquid medium with spectinomycin resistant TOP10 cells was done.
13 June 2018
Precipitate was detected in inoculated LB liquid medium.

LB liquid medium with spectinomycin was prepared.

Inoculation of the LB liquid medium with spectinomycin resistant TOP10 cells was done.
14 June 2018
MiraPrep was done.

Extracted plasmid samples were measured by Nanodrop.

Samples 260/280 260/230 Concentration

1 1.85 2.25 1915 ng/ul

Gel Electrophoresis was done.
15 June 2018
OD of liquid cyanobacteria culture was measured. OD: 1.035.
16 June 2018
OD of liquid cyanobacteria culture was measured. OD: 1.5.

Transformation of cyanobacteria (16/06/18, OD: 1.5) with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
17 June 2018
Transformed cyanobacteria (16/06/18) were plated onto BG-11 agar plates with spectinomycin.

WEEK 8 (June 18 – June 24)

18 June 2018
Extracted plasmid sample was linearized by digestion with EcoRI.

Gel Electrophoresis done.
19 June 2018
Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
20 June 2018
Transformed cyanobacteria (19/06/18) cells were plated onto BG-11 agar plates with spectinomycin.

BG-11 medium was prepared.

WEEK 9 (June 25 – July 1)

25 June 2018
BG-11 agar plates with spectinomycin were prepared.

OD of liquid cyanobacteria culture was measured. OD: 1.5.

Transformation of cyanobacteria (05/06/18, OD: 1.5) with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
26 June 2018
Transformed cyanobacteria (25/06/18) were plated onto BG-11 agar plates with spectinomycin.

Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
27 June 2018
Transformed cyanobacteria (26/06/18) cells were plated onto BG-11 agar plates with spectinomycin.

OD of liquid cyanobacteria culture was measured. OD: 1.680.

Transformation of cyanobacteria (05/06/18, OD: 1.680) with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.

New subculture of cyanobacteria was prepared from old liquid culture (05/06/18, OD: 1.680)
28 June 2018
Transformed cyanobacteria (27/06/18) cells were plated onto BG-11 agar plates with spectinomycin.

New subculture of cyanobacteria was prepared from old liquid culture (05/06/18).

WEEK 10 (July 2 – July 8)

2 July 2018
New subculture of cyanobacteria was prepared from old liquid culture (20/06/18).
3 July 2018
LB liquid medium and LB agar plates were prepared.

BG-11 medium was prepared.

New subculture of cyanobacteria was prepared from old liquid culture (20/06/18).
4 July 2018
BG-11 agar plates with spectinomycin were prepared.
5 July 2018
Two transformations of cyanobacteria (OD: 0.511 and OD: 0.948) with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) were done.
6 July 2018
Transformed cyanobacteria cells were plated onto BG-11 agar plates with spectinomycin.

Colony segregation was done (colony from 27/05/18 was streaked onto new plate).

WEEK 11 (July 9 – July 15)

9 July 2018
PCR amplification of SQR genes (Lep and Gei) was done.
10 July 2018
OD of liquid cyanobacteria culture (20/06/18) was measured. OD: 1.562.

Gel Electrophoresis with PCR products and Genes as control was run.
11 July 2018
Gel Electrophoresis with PCR products and Genes as control was run.

Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
12 July 2018
Transformed cyanobacteria cells were plated onto BG-11 agar plates with spectinomycin.

PCR purification was done for PRC products by using PCR purification kit. Concentration of SQR Geitlerinema (SQR Gei): 91.23, SQR Leptolyngbya (SQR Lep): 81.66, SuperNova: 50.59 ng/ul.

Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
13 July 2018
Transformed cyanobacteria cells were plated onto BG-11 agar plates with spectinomycin

WEEK 12 (July 16 – July 22)

17 July 2018
Interlab was started.

OD of liquid cyanobacteria culture (02/07/18) was measured. OD: 0.8.
18 July 2018
PCR amplification of SQR Gei was done again and no favorable results were obtained.

Gel Electrophoresis with PCR products was run.
19 July 2018
PCR amplification with restriction sites of Lep, Lep with signal sequence (ss), Gei and Gei with signal sequence (ss).

PCR purification was done for PRC products by using PCR purification kit.
20 July 2018
Lep, Lep with ss, Gei, Gei with ss and plasmid pSyn_6 were digested with BlII and KpnI restriction enzymes.

Gel extraction was done.

PCR amplification of Lep, Lep with ss, Gei and Gei with ss was done.
21 July 2018
Ligation of SQR genes (Lep, Lep with ss, Gei and Gei with ss) with pSyn_6 vector was done.

Transformation of E.coli (TOP10) with modified plasmid pSyn_6 (SQR genes and marker gene: spectinomycin resistance gene) was done.

Transformed E.coli cells were plated onto LB agar plates with spectinomycin.
22 July 2018
Inoculation of the LB liquid medium with spectinomycin of resistant TOP10 cells was done.

WEEK 13 (July 23 – July 29)

23 July 2018
Only transformed Gei E.coli grew up.

MiraPrep was done.

Extracted plasmid samples were measured by Nanodrop.

Samples 260/280 260/230 Concentration

Gei 1.98 2.35 410.5 ng/ul

Gel Electrophoresis was done. No bands.
24 July 2018
Competent E.coli (TOP10) cells were prepared.

OD of liquid cyanobacteria culture was measured.

Untransformed E.coli was plated on a plate with spectinomycin to check antibiotic activity.

25 July 2018

Untransformed E.coli grew up in plate with spectinomycin. It was deduced that antibiotic in plates was degraded.

Interlab was done.

PCR amplification of SQR genes with different annealing temperature (Lep and Gei) was done.

PCR purification was done for PRC products by using PCR purification kit.

PCR products were measured by Nanodrop.

Samples	260/280	260/230	Concentration
Lep (72C)	1.95	1.48	87.22 ng/ul
Lep (80C)	1.96	3.04	41.29 ng/ul
Gei (72C)	1.90	1.39	54.68 ng/ul
Gei (80C)	1.89	1.19	68.95 ng/ul

Lep, Lep with ss, Gei, Gei with ss and pSyn_6 plasmid were digested with BlII and KpnI restriction enzymes.

Gel extraction was done.

27 July 2018
The ligation of SQR genes (Lep, Lep with ss, Gei and Gei with ss) with pSyn_6 plasmid was done (1:10, insert : gene). Overnight incubation.

Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done.
28 July 2018
Transformed cyanobacteria (27/07/18) cells were plated onto BG-11 agar plates with spectinomycin.

Gel Electrophoresis of ligated pSyn_6 and SQR genes was run. SQR genes were not inserted.
23 July 2018
Ligation of SQR genes (Lep, Lep with ss, Gei and Gei with ss) with pSyn_6 plasmid was done (1:2, insert : gene). 2 hours incubation.

WEEK 14 (July 30 – August 5)

31 July 2018
Transformation of cyanobacteria with empty pSyn_6 vector (marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.

New subculture of cyanobacteria was prepared from old liquid culture.
1 August 2018
Transformed cyanobacteria (31/06/18) cells were plated onto BG-11 agar plates with spectinomycin.

PCR amplification of SQR genes was done.

Gel Electrophoresis of PCR products was run.
3 August 2018
Lep, Lep with ss, Gei, Gei with ss and plasmid pSyn_6 were digested with BlII and ScaI restriction enzymes.

Ligation of SQR genes (Lep, Lep with ss, Gei and Gei with ss) with pSyn_6 plasmid was done.

Transformation of E.coli (TOP10) with modified plasmid pSyn_6 (SQR genes and marker gene: spectinomycin resistance gene) was done.

Transformed E.coli cells were plated onto LB agar plates with spectinomycin.
4 August 2018
OD of liquid cyanobacteria culture was measured
5 August 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

WEEK 5

25 May 2018
OD of liquid cyanobacteria culture was measured

Samples	260/280	260/230	Concentration
1	1.76	1.53	63.96 ng/ul
2	1.96	2.29	1953 ng/u