Difference between revisions of "Team:Aachen/Parts"
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=Favorite Parts= | =Favorite Parts= | ||
* targeting plasmids [http://parts.igem.org/Part:BBa_K1585350 BBa_K1585350] [http://parts.igem.org/Part:BBa_K1585351 BBa_K1585351] | * targeting plasmids [http://parts.igem.org/Part:BBa_K1585350 BBa_K1585350] [http://parts.igem.org/Part:BBa_K1585351 BBa_K1585351] | ||
− | * [http://parts.igem.org/Part:BBa_K1585321 K1585321] | + | * [http://parts.igem.org/Part:BBa_K1585321 K1585321] polycistronic ''GlgCAB'' |
* polycistronic methanol assimilation pathway [http://parts.igem.org/Part:BBa_K1585241 BBa_K1585241] | * polycistronic methanol assimilation pathway [http://parts.igem.org/Part:BBa_K1585241 BBa_K1585241] | ||
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First, the different cells as well as their respective fragments and lysate supernatants were screened for formaldehyde production. The samples were taken 6 h after induction from shake flask cultures. Several cells, fragments and supernatants showed a production of formaldehyde but the Mdh activity in the intact cells was higher for each strain. | First, the different cells as well as their respective fragments and lysate supernatants were screened for formaldehyde production. The samples were taken 6 h after induction from shake flask cultures. Several cells, fragments and supernatants showed a production of formaldehyde but the Mdh activity in the intact cells was higher for each strain. | ||
− | The formaldehyde assay was repeated only with whole cells and additional samples taken | + | The formaldehyde assay was repeated only with whole cells and additional samples taken 20 h after induction. Moreover, the assay was conducted not only at 37 °C but also at 30 °C. The GlgC expressing BL21 Gold was used as a negative control. Again, in several strains functional Mdh could be detected and some general conclusions could be drawn: |
* More formaldehyde was produced in the samples taken 6 h after induction | * More formaldehyde was produced in the samples taken 6 h after induction | ||
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{{Team:Aachen/Figure|Aachen_15-07-09_glgB_expression_test_2.png|title=SDS-PAGE of GlgB in comparison to mRFP |subtitle=After induction with IPTG at OD=0.6, ''glgB'' was expressed. Samples were taken 6 and 19.5 hours after induction. The small arrows indicates the expected bands for GlgB. mRFP in pSB1K30 (T7 promoter) was used as negative control.|size=medium}} | {{Team:Aachen/Figure|Aachen_15-07-09_glgB_expression_test_2.png|title=SDS-PAGE of GlgB in comparison to mRFP |subtitle=After induction with IPTG at OD=0.6, ''glgB'' was expressed. Samples were taken 6 and 19.5 hours after induction. The small arrows indicates the expected bands for GlgB. mRFP in pSB1K30 (T7 promoter) was used as negative control.|size=medium}} | ||
+ | |||
+ | We wanted to investigate the functionality of the strains expression ''glgB''. Therefore, we used [[Team:Aachen/Notebook/Protocols#Dinitrosalicylic_Acid_Staining| dinitrosalicylic acid staining (DNS)]] for detection of reducing ends <ref>S. K. Meur, V. Sitakara Rao, and K. B. De. Spectrophotometric Estimation of Reducing Sugars by Variation of pH. Z. Anal. Chem. '''283''', 195-197 (1977)</ref> which should correspond to the branching frequency. All samples of ''glgB'' strains were compared to wild type samples. For best comparison the samples were grown to stationary phase and adjusted to the same OD before staining. Since every sugar or alkyl would have reacted with 3,5-dinitrosalicylic acid, we purified our samples before the staining. In order to identify the branching frequency, we analyzed the absorbance values of the purified samples compared to the absorbance values of hydrolyzed samples. By calculating the absorbance ratio of the non-hydrolyzed divided by the hydrolyzed samples, we aimed for information about the branches per glycogen unit. | ||
+ | The reaction principle can be described as follows. | ||
+ | |||
+ | {{Team:Aachen/Figure|Aachen_GlycogenDNS_assay_reaction_scheme.png|title=reaction principle|size=large}} | ||
+ | |||
+ | <span style="color:transparent">a</span> | ||
+ | |||
+ | 3,5-Dinitrosalicylic acid reacts with the reducing ends to 3-Amino-5-Nitrosalicylic acid, resulting in a changed π-system and therefore a change in absorbance. During the reaction, the reducing ends are oxidized whereas one nitro group of 3,5-Dinitrosalicylic acid is reduced to an amino group of 3-Amino-5-Nitrosalicylic acid. The more free reducing ends are present the more 3-Amino-5-Nitrosalicylic acid will be formed. Thus, the absorbance will increase. | ||
+ | |||
+ | |||
+ | ==Results from Dinitrosalicylic Acid Staining== | ||
+ | The absorbance of non-hydrolyzed samples of the ''glgB'' strain is higher than the absorbance of the wild type (see figure ''DNS staining of hydrolyzed samples of the ''glgB'' strain and wild type''). To identify the amount of glycogen in the samples, we hydrolyzed the samples and applied our staining. Our results show that the number of branches in glycogen is higher in the ''glgB'' strain compared to the wild type (''DNS staining of non-hydrolyzed samples of the ''glgB'' strain and wild type''). | ||
+ | |||
+ | |||
+ | |||
+ | {{Team:Aachen/DoubleFigure|Aachen GlycogenDNSNotHydrolyzed.png|Aachen GlycogenDNSHydrolyzed.png|title1=DNS staining of non-hydrolyzed samples of the ''glgB'' strain and wild type|title2=DNS staining of hydrolyzed samples of the ''glgB'' strain and wild type|subtitle1=The non-hydrolyzed samples of the ''glgB'' strain and wild type show that the overexpression of ''glgB'' leads to glycogen molecules with a higher number of branches compared to the wild type. Error bars show propagation of uncertainty.|subtitle2=The hydrolyzed samples indicate that the glycogen amount is higher in the wild type compared to the ''glgB'' overexpressing strain. Error bars show propagation of uncertainty. Since cell fragments are still left in the samples, the blank also shows an absorbance. Therefore, the line does not go though the origin.|size=medium}} | ||
+ | |||
+ | <span style="color:transparent">a</span> | ||
+ | |||
+ | The non-hydrolyzed values divided by the hydrolyzed values generate a ratio. Based on this ratio we can identify the number of branches per glycogen unit. It indicates that the number of branches per unit is higher in the ''glgB'' strain compared to the wild type. | ||
+ | |||
+ | |||
+ | {{Team:Aachen/DoubleFigure|Aachen GlycogenDNSRatio.png|Aachen_GlycogenDNSCalibration.png|title1=ratio of non-hydrolyzed and hydrolyzed DNS staining values of the ''glgB'' strain and wild type|title2=glycogen calibration curve|subtitle1=Although the amount of glycogen is higher in the wild type (see figure above), the number of branches per glycogen unit is higher in the ''glgB'' strain. Therefore the overexpression of ''glgB'' successfully increased the number of branches. Error bars show propagation of uncertainty.|subtitle2= With the calibration curve we are able to determine the glycogen concentration of our samples. Error bars show propagation of uncertainty.|size=medium}} | ||
==K1585312== | ==K1585312== | ||
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==K1585320== | ==K1585320== | ||
− | This composite part combines [http://parts.igem.org/Part:BBa_K1585320 | + | This composite part combines [http://parts.igem.org/Part:BBa_K1585320 glgA and glgB] for glycogen synthesis. GlgA uses ADP-glucose for chain elongation (α-1,4 linked) whereas GlgB uses existing chains to form α-1,6-linked branches. |
==K1585321== | ==K1585321== | ||
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The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of Bl21 Gold (DE3) transformants of BBa_K1585321 picture indicates considerably more glycogen accumulations compared to the wild type. | The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of Bl21 Gold (DE3) transformants of BBa_K1585321 picture indicates considerably more glycogen accumulations compared to the wild type. | ||
− | {{Team:Aachen/Figure| | + | {{Team:Aachen/Figure|Aachen_glgCAB_,_WT_v2.png|title=Iodine stained samples of Bl21 Gold (DE3) with glgCAB compared to the wild type|subtitle=The samples were taken from overnight cultures in LB + 20 mM glucose and were adjusted to the same OD before staining with 200 µl Lugol's iodine solution.|size=medium}} |
==K1585241== | ==K1585241== | ||
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After cloning both the targeting plasmid and the pCas plasmid in BL21 Gold (DE3) and inducing with arabinose, the Cas9 gene is expressed. Guided by the sgRNA, it cleaves the double strand at the beginning of the ''glgX'' gene. The succesful genome editing could be verified by sequencing. | After cloning both the targeting plasmid and the pCas plasmid in BL21 Gold (DE3) and inducing with arabinose, the Cas9 gene is expressed. Guided by the sgRNA, it cleaves the double strand at the beginning of the ''glgX'' gene. The succesful genome editing could be verified by sequencing. | ||
− | {{Team:Aachen/Figure|Aachen_glgX_knockout_sequencing. | + | {{Team:Aachen/Figure|Aachen_glgX_knockout_sequencing.JPG|title=Genomic region and sequencing chromatograms of BL21 Gold (DE3) Δ''glgX''|subtitle=The sequencing results show that the "IGEM AACHEN MULTISTOP" sequence disrupts the gene. This insert contains a stop codon in each possible frame. The picture demonstrates the functionality of BBa_K1585350.|size=large}} |
==K1585351== | ==K1585351== | ||
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{{Team:Aachen/Figure|Aachen_Bild_genome_edit_IGEM_AACHEN.jpg|title=Iodine staining of BL21 Gold (DE3) ''glgP'' knockout compared to BL21 Gold (DE3) wild type|subtitle=Iodine staining was performed with overnight cultures in M9 medium which were adjusted to the same OD. The darker color of the ''glgP'' knockout strain shows that more glycogen is present. Thus, the effectivity of the targeting plasmid is shown.|size=large}} | {{Team:Aachen/Figure|Aachen_Bild_genome_edit_IGEM_AACHEN.jpg|title=Iodine staining of BL21 Gold (DE3) ''glgP'' knockout compared to BL21 Gold (DE3) wild type|subtitle=Iodine staining was performed with overnight cultures in M9 medium which were adjusted to the same OD. The darker color of the ''glgP'' knockout strain shows that more glycogen is present. Thus, the effectivity of the targeting plasmid is shown.|size=large}} | ||
+ | |||
+ | =References= | ||
+ | <references /> | ||
{{Team:Aachen/Footer|color=purple}} | {{Team:Aachen/Footer|color=purple}} |
Latest revision as of 03:41, 19 September 2015