08.09.13

DH5α template was isolated from E.coli by Anatolia Geneworks isolation kit.

09.09.13

PCR of nhaA promoter region at different annealing temperatures (550-620C) by using of nhaA_fwd and nhaA_rvs primers.

10.09.13

Screening of PCR products with %1 Agrose Gel Electrophoresis. The best amplification was observed at 590C annealing temperature. Based on the agarose gel results, nhaA prometer region was amplified with 590C annealing temperature.

11.09.13

The promoter region was clonned and transformated to DH5α E.coli strain.

12.09.13

Single colonies obtained

13.09.13

Plasmid DNA was obtained from single colonies and was digested by EcoRI and SpeI

14.09.13

The enzyme digestion results were screened with %1 Agrose Gel Electrophoresis. The insert was not onserved on Agarose gel.

15.09.13

Enzyme digestion was set up again.

16.09.13

The enzyme digestion results were again screened , but this time it was studied with %3 Agrose Gel Electrophoresis. Unfortunately, No insert was observed.

17.09.13

PCR was performed again from DH5α template with nhaA promoter region primers.

18.09.13

Chloramphenicol vectors were extracted from Agarose Gel. PCR product (nhaA promoter region) was ligated to Chloramphenicol vector.

19.09.13

Transformation of ligation products were conducted.

20.09.13

Control of ligation. Store at +4.

22.09.13

3 colonies of ligation were selected and inoculated for overnight at 37 oC. However, we forgat to add antibiotic into our inoculated medium.

23.09.13

5 tubes plasmid isolation were conducted.

24.09.13

Enzyme digestion was performed by using of (EcoRI+PseI), (XbaI+PseI), EcoRI, PseI and XbaI enzymes.

27.09.13

The results of enzyme digestion was controlled with %1 Agrose Gel Electrophoresis.

28.09.13

3 colonies of ligation products were selected and inoculated for plasmid isolation. At that time, we did not forget to add antibiotic :)

29.08.2013

On the first day, parts that were used as weak constitutive promoter (18 G) and ribosome binding site (1I) were chosen. Also, these two parts were transformed to Escherichia coli and they were incubated overnight at 37 °C. In addition kanamycin plate was used for constitutive promoter and chloramphenicol plate was used for ribosome binding site part.

30.08.2013

Colonies were observed and single colony was chosen and inoculated in 5 mL LB medium to incubate overnight at 37 °C.

31.08.2013

From overnight bacterial culture, plasmids were isolated and plasmid concentrations were measured by nanodrop. Then, isopropanol precipitation was made in order to increase plasmid concentration. After this process, restriction reactions were set up for these two parts and ampicillin backbone. Plasmids that had 18 G were restricted with EcoRI and SpeI; also, plasmids that had 1I were restricted with XbaI and PtsI restriction enzymes. In addition ampicillin backbone was restricted with EcoRI and PtsI. Restriction conditions were 37 °C for 4 hours. At the end of the restriction reactions, they were incubated at 65 °C for 15 minutes in order to fix the enzymes. Finally, ligation reaction was set up in order to ligate promoter, ribosome binding site and ampicillin backbone. Also, it was incubated overnight at 4 °C.

01.09.2013

Colonies were observed and single colony was chosen in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C.

02.09.2013

From overnight bacterial culture, plasmids were isolated and plasmid concentrations were measured by nanodrop. Restriction reaction was set up again with EcoRI and PtsI at 37 °C for 4 hours. Then, products of restriction reaction and uncut plasmid (18G+ 1I with ampicillin backbone) were loaded into agarose gel. The gel was visualized and the sizes of the bands were compared with marker. Unfortunately, sizes of the bands were not correlated with our ligation product length.

05.09.2013

Our new gene fragment MazF was mixed with 20 µl distilled water. Also, ligation reactions were set up in order to ligate them into the cloning vector. Then, ligation products were transformed to E. coli. Moreover, terminator part (4F) was transformed and all of them were inoculated in agar plate.

06.09.2013

Colonies were observed and single colony was chosen from MazF and 4F plate in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C.

07.09.2013

From overnight bacterial culture, plasmids were isolated and plasmid concentrations were measured by nanodrop. According to measurements restriction reaction was set up. Plasmids that had MazF were restricted with EcoRI and SpeI, plasmids that had terminator were restricted with XbaI and PtsI restriction enzymes. Also, kanamycin backbone was restricted with EcoRI and PtsI enzymes. Finally, ligation reaction was set up in order to ligate MazF, terminator and kanamycin backbone. Also, it was incubated overnight at 4 °C.

08.09.2013

Other new gene fragments MazE, pLsrA and PTS p0 were mixed with 20 µl distilled water. Also, ligation reactions were set up in order to ligate them into the cloning vector. Then, ligation products were transformed to E. coli and they were incubated in ampicillin agar plate overnight at 37 °C.

09.09.2013

Colonies were observed and single colony was chosen in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C. In addition, red fluorescent protein (12N), 18 G and 1I were transformed again and inoculated in agar plate.

10.09.2013

From overnight bacterial culture, plasmids were isolated and plasmid concentrations were measured by nanodrop. According to measurements restriction reaction was set up. Plasmids that had all new gene fragments (MazE, MazF, pLsrA and PTS p0) were restricted with EcoRI and PtsI at 37 °C for 4 hours. After that process they were ligated with chloramphenicol backbone for 3 hours at 22 °C. Then, ligation products were transformed to E. coli and they were incubated in chloramphenicol agar plate overnight at 37 °C. Furthermore, colonies were observed in 12 N and 1I plates. Single colony was chosen in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C.

11.09.2013

From overnight bacterial culture, plasmids were isolated and plasmid concentrations were measured by nanodrop. According to measurements restriction reactions were set up. Plasmids that had 12 N were restricted with EcoRI and PtsI; plasmids that had 1I were restricted with XbaI and PtsI restriction enzymes. Then, 12 N was ligated with kanamycin backbone. Also, colonies were observed in agar plates that had ligation products of MazE, MazF, pLsrA and PTS p0 and single colonies were chosen in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C.

12.09.2013

From overnight bacterial culture, MazE, MazF, pLsrA and PTS p0 with chloramphenicol backbone plasmids were isolated and they were stored at -20 °C. Then, MazF and 4F plasmids that were obtained from cloning vector and they have ampicillin resistance were restricted with EcoRI and SpeI. Then, MazF was ligated with terminator (4F) and kanamycin backbone; also, PTS p0 was ligated with RBS (1I) and kanamycin backbone. Moreover, ligation products were transformed to E. coli and they were inoculated in agar plate overnight.

13.09.2013

Colonies were observed plate and single colony was chosen in order to inoculate LB medium. Moreover, it was incubated overnight at 37 °C. In addition, 18 G was transformed to E. coli again and inoculated in agar plate.

14.09.2013

From overnight bacterial culture, plasmids were isolated. Colonies were not observed in 18 G agar plate. Transformation of 18 G was made and it was inoculated in agar plates that had different antibiotics ampicillin, kanamycin and chloramphenicol.

15.09.2013

Single colony was taken form 18 G plate and incubated at LB medium overnight. Restriction reaction was set up for plasmids, which had 12 N, with EcoRI and SpeI enzymes. Then, products were ligated with terminator (4F) that was restricted with XbaI and PtsI before. Also, plasmids that had MazE were restricted with EcoRI and SpeI enzymes. Then, products were also ligated with terminator (4F) overnight at 4 °C.

16.09.2013

Ligation products were transformed to bacteria and they were inoculated in agar medium overnight. Unfortunately, there was no proliferation in LB medium. Transformation of 18 G part was not achieved.

17.09.2013

Colonies were observed and single colonies were taken in order to inoculate in LB medium. They were incubated overnight.

18.09.2013

From overnight bacterial culture, plasmids were isolated. Then, plasmids that had PTS p0 and 1I were restricted with EcoRI and SpeI enzymes. Also, plasmids that had MazE and 4F were restricted with XbaI and PtsI I enzymes. Moreover, plasmids that had 12N and 4F were restricted with XbaI and PtsI I enzymes. By using products of these reactions, two ligation reactions were set up at 4 °C overnight. PTS p0 (promoter) and 1I (RBS) gene parts were ligated with MazE and 4F (terminator) gene parts with chloramphenicol backbone. Also, PTS p0 (promoter) and 1I (RBS) gene parts were ligated with 12N (RFP) and 4F (terminator) gene parts in chloramphenicol backbone.

19.09.2013

Ligation products were transformed to E. coli and they were inoculated in agar plate overnight.

20.09.2013

Transformations of ligation products were not achieved. Thus, ligation reactions were set up again. PTS p0 (promoter) and 1I (RBS) gene parts were ligated with MazE and 4F (terminator) gene parts with chloramphenicol backbone. Also, PTS p0 (promoter) and 1I (RBS) gene parts were ligated with 12N (RFP) and 4F (terminator) gene parts in chloramphenicol backbone.

21.09.2013

Ligation products were transformed to E. coli and they were inoculated in agar plate overnight.

22.09.2013

Transformations of ligation products were not achieved. In order to isolate PTS p0 (promoter), 1I (RBS), MazE, 12 N (RFP) and 4F (terminator), cells were inoculated in LB medium overnight.

23.09.2013

From overnight bacterial culture, plasmids were isolated. High concentration of PTS p0 (promoter), 1I (RBS), MazE and 4F (terminator) plasmids were obtained. Then, restriction reactions were set up again. Plasmids that contained PTS p0, MazE and 12 N (RFP) were restricted with EcoRI and SpeI enzymes. Also, 1I (RBS) and 4F (terminator) were restricted with XbaI and PseI I enzymes. Then, MazE was ligated with terminator (4F) and ampicillin backbone; also, PTS p0 was ligated with RBS (1I) and kanamycin backbone. Moreover, 12 N (RFP) was ligated with 4 F and ampicillin backbone.

24.09.2013

Ligation products were transformed to E. coli and they were inoculated in agar plate overnight.

25.09.2013

Colonies were observed and single colonies were taken in order to inoculate in LB medium. They were incubated overnight.

26.09.2013

From overnight bacterial culture, plasmids were isolated. Plasmids that had PTS p0 and RBS (1I), plasmids that had 12 N (RFP) and terminator (4F); and also, plasmids that had MazE and and terminator (4F) were obtained. Ligation reactions were set up again. PTS p0 (promoter) and 1I (RBS) gene parts were ligated with MazE and 4F (terminator) gene parts with chloramphenicol backbone. Also, PTS p0 (promoter) and 1I (RBS) gene parts were ligated with 12N (RFP) and 4F (terminator) gene parts in chloramphenicol backbone.

27.09.2013

Ligation products were transformed to E. coli and they were inoculated in agar plate overnight.

28.09.2013

Colonies were observed and single colonies were taken in order to inoculate in LB medium. They were incubated overnight.