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

July

Week 10-13

Week 10	July 2 – July 8
Week 11	July 9 – July 15
Week 12	July 16 – July 22
Week 13	July 23 – July 29

August

Week 14-18

Week 14	July 30 – August 5
Week 15	August 6 – August 12
Week 16	August 13 – August 19
Week 17	August 20 – August 26
Week 18	August 27 – September 2

September

Week 19-22

Week 19	September 3 – September 9
Week 20	September 10 – September 16
Week 21	September 17 – September 23
Week 22	September 24 – September 30

October

Week 23-25

Week 23	October 1 – October 7
Week 24	October 8 – October 14
Week 25	October 15 – October 21

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 (72℃)	1.95	1.48	87.22 ng/ul
Lep (80℃)	1.96	3.04	41.29 ng/ul
Gei (72℃)	1.90	1.39	54.68 ng/ul
Gei (80℃)	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
All plates had colonies except the plate with Lep + pSyn_6 E.coli (TOP10).

Inoculation of the LB liquid medium with spectinomycin resistant TOP10 cells was done (Lep with ss, Gei and Gei with ss).

5 August 2018

MiraPrep of Gei, Gei with ss and Lep with ss was done.

Extracted plasmid samples were measured by Nanodrop

Samples	260/280	260/230	Concentration
Gei	1.88	2.24	2278 ng/ul
Gei with ss	1.86	2.26	1893 ng/ul
L with ss (1)	1.87	2.20	2787 ng/ul
L with ss (2)	1.84	2.28	2518 ng/ul

WEEK 15 (August 6 – August 12)

6 August 2018
Extracted plasmid samples (Gei, Gei with ss and Lep with ss) were linearized by digestion with ScaI.

Gel Electrophoresis with linearized Gei, Gei with ss and Lep with ss (1 and 2) was run.
7 August 2018
Digestion-ligation procedure was repeated with Lep gene.

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

Transformed E.coli cells were plated onto LB agar plates with spectinomycin.
8 August 2018
Inoculation of the LB liquid medium with spectinomycin of resistant TOP10 cells was done (Lep).
9 August 2018
MiraPrep of Lep was done.

Extracted plasmid sample were measured by Nanodrop.

Samples 260/280 260/230 Concentration

Lep 1.86 2.24 3053 ng/ul
10 August 2018
Another lamp with high light intensity was tested for cyanobacteria incubation.

WEEK 16 (August 13 – August 19)

15 August 2018
Transformation of cyanobacteria with modified plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
16 August 2018
Transformed cyanobacteria (15/08/18) cells were plated onto BG-11 agar plates with spectinomycin.

Transformation of cyanobacteria with modified plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
17 August 2018
Transformed cyanobacteria (16/08/18) cells were plated onto BG-11 agar plates (gradient) with spectinomycin.
18 August 2018
New subculture of cyanobacteria was prepared from old liquid culture.

OD of liquid cyanobacteria culture was measured.

WEEK 17 (August 20 – August 26)

20 August 2018
Plates with new transformations were checked.
21 August 2018
Plates with new transformations were checked. Stock solutions for next lab procedures were prepared
23 August 2018
OD of new liquid cyanobacteria culture was measured.
26 August 2018
Gei, Gei with ss, Lep and Lep with ss plasmid samples were linearized by digestion with ScaI.

Gel Electrophoresis with linearized Gei, Gei with ss, Lep and Lep with ss was run.

WEEK 18 (August 27 – September 2)

27 August 2018
Transformation of cyanobacteria with modified plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
28 August 2018
Transformed cyanobacteria (27/08/18) cells were plated onto BG-11 agar plates (with NaHCO₃ and without) with spectinomycin.
29 August 2018
Gei, Gei with ss, Lep and Lep with ss plasmid samples were linearized by digestion with ScaI.

Gel Electrophoresis with linearized Gei, Gei with ss, Lep and Lep with ss was run.

Transformation of cyanobacteria with modified linear plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
30 August 2018
Transformed cyanobacteria (29/08/18) cells were plated onto BG-11 agar plates with spectinomycin.
1 September 2018
New subculture of cyanobacteria was prepared from old liquid culture.

OD of new liquid cyanobacteria culture was measured.

Transformation of cyanobacteria with modified linear plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
2 September 2018
Transformed cyanobacteria (01/09/18) cells were plated onto BG-11 agar plates with spectinomycin.

WEEK 19 (September 3 – September 9)

3 September 2018
Gei, Gei with ss, Lep and Lep with ss plasmid samples were linearized by digestion with ScaI.

Gel Electrophoresis with linearized Gei, Gei with ss, Lep and Lep with ss (1 and 2) was run.

Transformation of cyanobacteria with modified linear plasmid pSyn_6 (Gei, Gei with ss, Lep, Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
4 September 2018
Transformed cyanobacteria (03/09/18) cells were plated onto BG-11 agar plates with spectinomycin.

PCR amplification of empty pSyn_6 plasmid and modified pSyn_6 plasmid (Lep with ss) with Lep with ss primers.

Gel Electrophoresis of PCR products was run.
5 September 2018
Survival test of cyanobacteria with Na₂S under anaerobic conditions was done.
6 September 2018
Lamp with high light intensity was installed on plates with cyanobacteria
8 September 2018
All plates with cyanobacteria were checked

WEEK 20 (September 10 – September 16)

10 September 2018
Colony PCR was done with cyanobacteria colonies.

Gel Electrophoresis of PCR products was run.
11 September 2018
Gel Electrophoresis of PCR products (10/09/18) was repeated.

Colony PCR was done with cyanobacteria colonies from different plates.

Gel Electrophoresis of PCR products (11/09/18) was run.
12 September 2018
Inoculation of the BG-11 medium with NaHCO₃ of cyanobacteria colonies from the plate was done.

Transformation of cyanobacteria with modified linear plasmid pSyn_6 (Lep with ss and marker gene: spectinomycin resistance gene) was done. Stony Brook University protocol.
13 September 2018
BG-11 agar plates with spectinomycin were prepared.

Transformed cyanobacteria (12/09/18) cells were plated onto BG-11 agar plates with spectinomycin.

Original pSyn_6 plasmid (empty) was tested for the presence of Lep gene through PCR and Gel Electrophoresis.
14 September 2018
Inoculation of the BG-11 medium with NaHCO₃ of cyanobacteria colonies from the plate was done.

Competent E.coli (DH5alpha) cells were prepared.

Transformation of E.coli (DH5alpha and TOP10) with original pSyn_6 plasmid (marker gene: spectinomycin resistance gene) was done.

Transformed E.coli cells were plated onto LB agar plates with spectinomycin.
15 September 2018
Inoculation of the LB liquid medium with spectinomycin of resistant DH5alpha and TOP10 cells was done.

16 September 2018

MiraPrep was done.

Extracted plasmid samples were measured by Nanodrop.

Samples	260/280	260/230	Concentration
TOP10	1.87	2.33	2319 ng/ul
DH5alpha	2.02	2.15	1200 ng/ul

WEEK 21 (September 17 – September 23)

17 September 2018
Extracted plasmid sample (16/09/18) was linearized by digestion with EcoRI.

Gel Electrophoresis was run.
18 September 2018
The pH of BG-11 medium with NaHCO₃ was adjusted before inoculation with cyanobacteria colonies.

Extracted plasmid sample (16/09/18) was tested for the presence of Lep gene through PCR and Gel Electrophoresis.
19 September 2018
Colony PCR of cyanobacteria colonies from the plate was done.

Gel Electrophoresis of PCR products was run.
20 September 2018
Colony PCR of cyanobacteria was done.

Gel Electrophoresis of PCR products was run.
21 September 2018
Liquid culture colony PCR of cyanobacteria was done.

Gel Electrophoresis of PCR products was run.
23 September 2018
Gel Electrophoresis of PCR products (20/09/18 and 21/09/18) was run.

WEEK 22 (September 24 – September 30)

25 September 2018
Colony PCR of cyanobacteria was done.

Gel Electrophoresis of PCR products was run.

Absorbance of Na₂S solution was measured and tested by Nanodrop.
26 September 2018
Colony PCR of cyanobacteria colonies was done. Different annealing temperatures were used.

Gel Electrophoresis of PCR products.
27 September 2018
Colony PCR of cyanobacteria colonies was done.

Gel Electrophoresis of PCR products was run
28 September 2018
Colony PCR of cyanobacteria colonies was done.

Gel Electrophoresis of PCR products was run.

New subculture of cyanobacteria was prepared from old liquid culture.

WWEEK 23 (October 1 – October 7)

1 October 2018
Colony PCR of cyanobacteria colonies (Lep with ss and without sqr gene) was done.

Gel Electrophoresis of PCR products was run.
4 October 2018
Liquid culture colony PCR of cyanobacteria was done.

Gel Electrophoresis of PCR products was run
5 October 2018
PCR amplification of SQR gene and Supernova with specific sequence for CPEC was done.

Gel Electrophoresis of PCR products was run.
6 October 2018
PCR amplification of SQR gene and Supernova with specific sequence for CPEC was done
7 October 2018
Gel Electrophoresis of PCR products (06/10/18) was run.

CPEC assembly of SQR gene and Supernova was done.

Gel electrophoresis was run.

Transformation of E.coli (TOP10) with modified plasmid pSB1C3 (SQR gene, Supernova and marker gene: chloramphenicol resistance) was done.

Transformed E.coli cells were plated onto LB agar plates with chloramphenicol.

WEEK 24 (October 8 – October 14)

8 October 2018
LB liquid medium with chloramphenicol was prepared.

Inoculation of the LB liquid medium with chloramphenicol of resistant TOP10 cells was done
9 October 2018
MiraPrep was done.

Gel Electrophoresis of extracted SQR and Supernova was run
10 October 2018
All DNA’s were submitted.
11 October 2018
Colony PCR of cyanobacteria was done

Gel Electrophoresis of PCR products was run
12 October 2018
Na₂S was dissolved in the BG-11 medium and after that liquid cyanobacteria culture was added. The concentration of Na₂S was measured after some period of time to analyse the activity SQR gene in cyanobacteria.
13 October 2018
Liquid cyanobacteria culture was tested on survival in oily water.

Na₂S reduction assay with cyanobacteria was done.
October 2018
New subculture of cyanobacteria was prepared from old liquid culture.

Na₂S reduction assay with cyanobacteria was done.

WEEK 25 (October 15 – October 21)

15 October 2018
Na₂S reduction assay with cyanobacteria was done
15 October 2018
Na₂S reduction assay with cyanobacteria was done

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