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Team:ITU MOBGAM Turkey/labdiary
From 2013.igem.org
Laboratory Diary
28.08.13
RBS (B0034) and T7 Promoter (I719005) were transformed to E.coli GM2163 competent cells, and inoculated in Apm Petri dishes to incubation for 16 hours at 37ºC.
29.08.13
There were no any colonies on the Petri dishes inoculated night before. GIF, RBS, and T7 Promoter were transformed. GIF was inoculated in Amp, others in Amp, CM, and Kan. Terminator (B0015) was inoculated into liquid Amp containing LB Medium and incubated for 16 hours, 37 º C at shaker.
30.08.13
Single colonies were observed in GIF, and smear was other ones. Thus, the Petri dishes were saved at 4 ºC. Both manual and Roshe plasmid isolation kit was applied to isolate the Terminator. However, low concentration and high impurity were obtained. Inoculation for liquid medium was made again for Terminator. T7 Promoter and RBS were inoculated by streak plate method to obtain single colony. Single colony from GIF was chosen and inoculated both liquid and solid Amp media.
31.08.13
GIF and Terminator were centrifuged and cells were saved at -20 ºC as stock. Single colonies were chosen from both RBS and T7 Promoter, and inoculated liquid and solid media.
01.09.13
Plasmid isolation was made to RBS and T7 Promoter by the kit and manually.
Double restriction digestion was applied both GIF and Terminator as follows:
02.09.13
Digestion samples and their uncut plasmids were run on the 1% Agarose gel electrophoresis. GIF was observed as expected unlike the Terminator. New single colony from the Terminator was inoculated both in liquid and solid CM containing media.
03.09.13
Terminator, T7 promoter, and RBS plasmids were run on the 1% Arasore gel. There were no any bands. Triple colonies were chosen from both RBS and T7 Promoter, and inoculated liquid and solid media. GIF was inoculated for the stock preparation. GIF was digested with EcoRI and PstI to insert into the CM vector.
04.09.13
Uncut, one-cut, and two-cut GIF were run on the 1% Agarose gel. All bands were observed as they expected.
Manual plasmid isolation was made for T7 Promoter and RBS. Plasmids were run on the gel.
Ligation was made as follows:
Terminator was inoculated by streak plate method from main stock to obtain single colony.
05.09.13
Ligations were transformed and inoculated on solid media. T7 Promoter, RBS, and Terminator were diluted as the 1:20 ratio, and transformation was made with these new ratios.
06.09.13 (Bad Friday)
E.coli strain was changed from GM2163 to DH5α and inoculation was made on solid medium.
Single colonies were chosen from GIF in CM Vector and GIF+Terminator in Kan vector plates and inoculated in liquid medium.
07.09.13 Plasmid isolation from GIF in CM Vector and GIF+Terminator in Kan vector were made and run on the Agarose gel. No band formation was observed.
DH5α transformation was made for GIF, Terminator, T7 Promoter, and RBS. Inoculation on solid media was made as follows:
GIF in Amp, Terminator in half-CM and CM, T7 Promoter in half-Amp and Amp, RBS in half-Amp and Amp, and DH5α in half-Amp, Amp, half-CM, CM, and Kan.
08.09.13 DH5α competent cells were prepared for -80ºC stock.
RBS, T7 Promoter, and Terminator were transformed into newly prepared competent cells.
Both transformations and positive and negative controls for DH5α were inoculated on the solid media.
GIF inoculated into both solid and liquid media.
09.09.13 T7 Promoter was transformed again, and inoculated to the half-Amp and Amp plates.
Plasmid isolation was made from GIF, and run on the Agarose gel.
10.09.13 Plasmid isolation was made from RBS and Terminator.
1:20 diluted T7 Promoter transformation was made, and inoculated.
Terminator and GIF were digested both single and double restriction digestion.
11.09.13 Plasmid isolation was made from RBS. There were no any bands.
Ligation was made as follows:
12.09.13 Competent cells were prepared, and both positive and negative controls were made.
Ligations were transformed and inoculated on the solid media.
15.09.13 Ligation was made as follows:
Ligations were transformed and inoculated on the solid media.
RBS and T7 Promoter were cleaned-up and transformed to be inoculated on solid media.
16.09.13 Triple colonies were chosen from GIF in CM Vector, GIF+Terminator in Kan vector, RBS, and T7 Promoter, and inoculated in both solid and liquid media.
17.09.13 Plasmid isolation was made from GIF in CM Vector, GIF+Terminator in Kan vector, and RBS. Agarose gel was applied to these plasmids.
Restriction digestion was made as follows:
18.09.13 oth uncut and cut plasmids were run on the Agarose gel.
Restriction digestion was made for GIF+Terminator in Kan vector for 3 hours at 37ºC as follows:
Plasmid isolation from RBS was made by kit and manually.
19.09.13 RBS and digestion plasmids run on Agarose gel.
20.09.13 Ligation was made for GIF+Terminator to insert into RBS vector with different vector: insert ratios as follows:
21.09.13 Ligations were transformed to competent cells and inoculated to Amp plate.
T7 Promoter was also inoculated to the Amp plate.
25.09.13 Double digestion was made for GIF+Terminator in RBS Vector as follows:
GIF+Terminator in RBS Vector was inoculated in both liquid and solid media.
RBS and GIF+Terminator in Kan Vector were inoculated in liquid media.
02.09.13
The vector, Pgex, transformed to the competent cells which have been already prepared and transformed cells were incubated into an agar plate that contains ampicillin for overnight.
03.09.13
Primers were diluted and stocked. The pcr reaction whose total volume was 25µl was prepared for GIF gene for 3 hours. Selected colonies from the agar plate were incubated into 5ml of an LB medium for overnight.
04.09.13
The pcr results were loaded wells of agarose gel and electrophoresis were started. The results were observed that were not clean because of that decided to make gel extraction. The plasmid isolation protocol was applied for cells which had incubated in LB medium and rate of OD was measured by nano-drop machine. The cleavage reaction was prepared for GIF gene and Pgex plasmids using enzymes, BamHI and XhoI.
This reaction occurred at 370C for 4 hours and then at 650C for 15 minutes.
05.09.13
Cleavage results were observed after the electrophoresis but there was no band for plasmids. Because of that, to isolation cell incubation was repeated. The multiplied GIF gene was not enough for the new cleavage reaction, so the PCR reaction was repeated.
06.09.13
The pcr results were observed on the gel and gel extract procedure was applied. Plasmids were isolated but the gel results showed that there was no plasmid and so that the incubation was repeated.
07.09.13
Plasmids were isolated but there was a few plasmids observed that was not enough OD value for cleavage reaction. The incubation of the cells was repeated.
08.09.13
Plasmids were isolated from the cells and the cleavage reaction was prepared for plasmid and GIF gene.
09.09.13
The cleavage products observed on the agarose gel and gel extraction was applied. Ligation reaction was prepared for Gıf gene and Pgex vector using T4 DNA ligase enzyme at 4oC for overnight.
10.09.13
The agarose gel electrophoresis procedure was applied for ligation products and they were incubated into agar plate that contained AMP.
11.09.13
There was only one colony on the plate and that colony was incubated into 5ml of LB growth that contained AMP.
12.09.13
It was observed that there was no growth in the LB medium. There were not enough PCR product and isolated plasmid for reactions because of that the PCR reaction was repeated for GIF gene and incubation of cells to isolation of Pgex vector was repeated.
13.09.13
Plasmid isolation was done. Isolated plasmids were uploaded into the agarose gel to observ. They had very high concentration so diluted with EB buffer. PCR products also observed by using electrophoresis and then gel extraction was applied.
14.09.13
Eccording to the OD value of plasmids, cleavage reaction was prepared for isolated plasmids and GIF gene that was prolifered by pcr protocol. Cleavage reaction: 1.rxn 2.rxn 3.rxn DNA 0.8 0.8 0.8 XhoI 1.2 1.2 - BamHI 1.2 - 1.2 Buffer B 5 5 5 BSA 0.5 0.5 0.5 Water 41.30 42.50 42.50 Reaction occurs at 37oC for 3 hours and then 65oC for 15 minutes. Cleavage results were observed on agarose gel and gel extraction was applied for double digestions.
15.09.13
Ligation reaction was prepared to ligate Pgex vector and GIF gene as an insert. Ligation reaction: Rxn1 Rxn2 Rxn3 DNA 0.46 0.33 0.2 GIF 2 2.8 4.6 T4 ligase 0.6 0.6 0.6 Buffer 2 2 2 Water 14.94 14.27 12.6 Incubated at 4oC for overnight.
16.09.13
Ligation results were uploaded into the agarose gel and observed. Extraction was done for results and they transferred to the E. coli. After transforrmation the cells were inocukated on to the agar plate that contained ampicillin.
17.09.13
The products of transformation did not grow because of that ligation reactions were repeated.
18.09.13
Ligation products were transformed to the bacteria and they were incubated on to the agar plate contained ampicillin.
19.09.13
It was observed that colonies were on the plate. To control the products are true or not, one colony was selected and incubated into LB medium and agar plate.
20.09.13
Plasmids were isolated from the E.coli cells. The dounle and single cleavage reactions were prepared.
21.09.13
Results of cleavage reaction were uploaded into the agarose gel. It was observed that reaction did not occur and the cleavage reaction was repeated.
22.09.13
The products of reaction were observed after the gel electrophoresis. The observation results showed that cleavage reaction was occured and also the plasmid contained GIF gene.
23.09.13
The cells that contained pgex with gif gene were incubated in LB medium.
24.09.13, 25.09.13
Single colony was selected from the agar plate and it was incubated into 25ml of LB medium.
26.09.13
Incubated cells were transferred to 250ml of LB medium and OD value was measured and then IPTG was added. After 3 hours, the cells were precipitated.
27.09.13
The binding buffer and elution buffer were prepared.
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.
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