Difference between revisions of "Team:Aachen/Lab/Glycogen/Characterization"
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After building our mono- and polycistronic constructs, their characterization was the next and possibly most important step. In this process, we investigated both cell growth and glycogen formation to prove the accumulation of glycogen due to the overexpression of glycogen building genes and the knockout of glycogen degradation genes. | After building our mono- and polycistronic constructs, their characterization was the next and possibly most important step. In this process, we investigated both cell growth and glycogen formation to prove the accumulation of glycogen due to the overexpression of glycogen building genes and the knockout of glycogen degradation genes. | ||
− | In our approaches, we wanted to examine the function of ''glgCAB'', draw conclusions on fermentation conditions and estimate the increase in glycogen. Therefore, we did growth experiments with ''Escherichia coli'' BL21 Gold (DE3) and analysed respective samples via [[Team:Aachen/Notebook/Protocols#Iodine_Staining|iodine staining]] and [[Team:Aachen/Notebook/Protocols#Dinitrosalicylic_Acid_Staining| dinitrosalicylic acid staining]]. Furthermore, we did glycogen purifications to apply a [[Team:Aachen/Notebook/Protocols#Glycogen_Kit|glycogen kit]]. | + | In our approaches, we wanted to examine the function of ''glgCAB'', draw conclusions on fermentation conditions and estimate the increase in glycogen. Therefore, we did growth experiments with ''Escherichia coli'' BL21 Gold (DE3) and analysed respective samples via [[Team:Aachen/Notebook/Protocols#Iodine_Staining|iodine staining]] and [[Team:Aachen/Notebook/Protocols#Dinitrosalicylic_Acid_Staining| dinitrosalicylic acid staining]]. Furthermore, we did glycogen purifications to apply a [[Team:Aachen/Notebook/Protocols#Glycogen_Kit|glycogen kit]]. |
− | + | ||
+ | ==Achievements== | ||
{|class="wikitable" | {|class="wikitable" | ||
! Part !! Gene !! '''Result ''' | ! Part !! Gene !! '''Result ''' | ||
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585321 K1585321]|| ''glgCAB'' || characterized and therefore | + | |[http://parts.igem.org/Part:BBa_K1585321 K1585321]|| ''glgCAB'' || characterized by iodine staining and therefore improved [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585320 K1585320]|| ''glgAB'' || characterized polycistronic BioBrick | + | |[http://parts.igem.org/Part:BBa_K1585320 K1585320]|| ''glgAB'' || characterized polycistronic BioBrick by iodine staining |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585310 K1585310]|| ''glgA'' || characterized monocistronic BioBrick | + | |[http://parts.igem.org/Part:BBa_K1585310 K1585310]|| ''glgA'' || characterized monocistronic BioBrick by iodine staining |
|- | |- | ||
− | |[http://parts.igem.org/Part: | + | |[http://parts.igem.org/Part:BBa_K1585311 K1585311]|| ''glgB'' || characterized monocistronic BioBrick by dinitrosalicylic acid staining |
|- | |- | ||
+ | |[http://parts.igem.org/Part:BBa_K1585351 K1585351]|| Δ''glgP'' || characterized gene knockout by iodine staining | ||
|} | |} | ||
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==Results from iodine staining== | ==Results from iodine staining== | ||
− | Our first BioBrick that was successfully characterized by iodine staining is the glycogen synthase, GlgA. The cell pellet of GlgA turned dark brown when resuspened in iodine solution while the wild type cells changed to a light yellow. The iodine molecules interact with the helical structure in the | + | Our first BioBrick that was successfully characterized by iodine staining is the glycogen synthase, GlgA. The cell pellet of GlgA turned dark brown when resuspened in iodine solution while the wild type cells changed to a light yellow. The iodine molecules interact with the helical structure in the glycogen and thereby change their absorbance and appear dark blue or black/brown. Therefore the dark color shows that more glycogen is present in the BL21 Gold (DE3) + ''glgA'' cells compared to the wild type. |
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− | Finally, after our polycistronic construct ''glgCAB'' was assembled in an expression vector, it was also cultivated on LB with 20 mM glucose and stained. In several trials, ''glgCAB'' was darker than the wild type. Compared to the already existing BioBrick (BBa_K118016)'' glgC'', our polycistronic construct was colored more brown than blue. This results from a higher braching frequency in the glycogen structure. This indicates that the branching enzyme in our ''glgCAB'' is, in spite of a point mutation, functionally expressed and influences the glycogen structure. | + | Finally, after our polycistronic construct ''glgCAB'' was assembled in an expression vector, it was also cultivated on LB with 20 mM glucose and stained. In several trials, ''glgCAB'' was darker than the wild type. Compared to the already existing BioBrick (BBa_K118016)'' glgC'', our polycistronic construct was colored more brown than blue. This results from a higher braching frequency in the glycogen structure.<ref> Lengeler J., Drews G., Schlegel H. Biology of the Prokaryotes, page 194, 9.5.2</ref>This indicates that the branching enzyme in our ''glgCAB'' is, in spite of a point mutation, functionally expressed and influences the glycogen structure. |
{{Team:Aachen/Figure|Aachen_glgCAB , WT_v2.png|title=Iodine staining BL21 Gold (DE3) + ''glgCAB'' vs. wild type |subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + ''glgCAB'' stained distinctly darker than the BL21 Gold (DE3) wild type.|size=medium}} | {{Team:Aachen/Figure|Aachen_glgCAB , WT_v2.png|title=Iodine staining BL21 Gold (DE3) + ''glgCAB'' vs. wild type |subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + ''glgCAB'' stained distinctly darker than the BL21 Gold (DE3) wild type.|size=medium}} | ||
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{{Team:Aachen/Figure|Aachen_15-09-17 deltaP +glgCAB vs deltaP.jpg|title=Iodine staining BL21 Gold (DE3) Δ''glgP'' + ''glgCAB'' vs. BL21 Gold (DE3) Δ''glgP''|subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) Δ''glgP'' + ''glgCAB'' stained distinctly darker than BL21 Gold (DE3) Δ''glgP''. It shows that even higher glycogen accumulation can be achieved by combining overexpression of all three synthesis enzymes and the ''glgP'' knockout.|size=medium}} | {{Team:Aachen/Figure|Aachen_15-09-17 deltaP +glgCAB vs deltaP.jpg|title=Iodine staining BL21 Gold (DE3) Δ''glgP'' + ''glgCAB'' vs. BL21 Gold (DE3) Δ''glgP''|subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) Δ''glgP'' + ''glgCAB'' stained distinctly darker than BL21 Gold (DE3) Δ''glgP''. It shows that even higher glycogen accumulation can be achieved by combining overexpression of all three synthesis enzymes and the ''glgP'' knockout.|size=medium}} | ||
− | |||
=Dinitrosalicylic Acid Staining= | =Dinitrosalicylic Acid Staining= | ||
− | The | + | The iodine staining of strains containing ''glgB'' (branching enzyme) was not quite distinct. We still wanted to investigate the functionality of the strains. 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> | ||
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+ | 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'' overexpressing 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 identify the glycogen concentration of our samples. Error bars show propagation of uncertainty.|size=medium}} | ||
+ | |||
+ | <span style="color:transparent">a</span> | ||
+ | |||
+ | |||
+ | The results of our experiments show that the overexpression of ''glgB'' leads to glycogen molecules with a higher number of branches compared to the wild type. Therefore, we proved the functionality of our ''glgB'' BioBrick. | ||
+ | |||
+ | <span style="color:transparent">a</span> | ||
=Growth experiments= | =Growth experiments= | ||
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==HPLC analysis== | ==HPLC analysis== | ||
− | Another approach to measure the glycogen accumulation quantitatively would be the analysis of samples by HPLC. With this method purified glycogen can be directly measured or the glucose levels can be observed after glycogen hydrolysis. We worked on this approach but the main problem was that we did not have a reliable glycogen purification protocol. Hence our samples could not be analyzed properly. Nevertheless, by optimizing this aspect the glycogen content of cells should be clearly visible as we have already seen correct peaks for the synthesis operon in the glgP knockout strain. | + | Another approach to measure the glycogen accumulation quantitatively would be the analysis of samples by[[Team:Aachen/Notebook/Protocols#HPLC| HPLC]]. With this method purified glycogen can be directly measured or the glucose levels can be observed after glycogen hydrolysis. We worked on this approach but the main problem was that we did not have a reliable glycogen purification protocol. Hence, our samples could not be analyzed properly. Nevertheless, by optimizing this aspect the glycogen content of cells should be clearly visible as we have already seen correct peaks for the synthesis operon in the ''glgP'' knockout strain. |
<span style="color:transparent">a</span> | <span style="color:transparent">a</span> | ||
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− | Over the course of our project, we initially pursued working in the subgroups “methanol” and “glycogen”. As our work progressed and first milestones were reached, we took a step forward and thought of ways to combine both aspects –methanol assimilation and glycogen accumulation- in one organism. Since the ''glgP'' single knockout in Bl21 Gold (DE3) gave good results regarding its growth performance and glycogen accumulation, we decided to transform the polycistronic construct of methanol assimilating genes (''mdh'',''hps'', ''phi'', ''xpk'') into this strain. We saw that this strain was able to grow on M9 with 0.522 M methanol. The most effective way of further optimizing its growth and methanol assmilitation | + | Over the course of our project, we initially pursued working in the subgroups “methanol” and “glycogen”. As our work progressed and first milestones were reached, we took a step forward and thought of ways to combine both aspects –methanol assimilation and glycogen accumulation- in one organism. Since the ''glgP'' single knockout in Bl21 Gold (DE3) gave good results regarding its growth performance and glycogen accumulation, we decided to transform the polycistronic construct of methanol assimilating genes (''mdh'',''hps'', ''phi'', ''xpk'') into this strain. We saw that this strain was able to grow on M9 with 0.522 M methanol. The most effective way of further optimizing its growth and methanol assmilitation is to apply promoters with different strengths. |
==[[Team:Aachen/Notebook/Documentation/Glycogen_Characterization|Laboratory Notebook]]== | ==[[Team:Aachen/Notebook/Documentation/Glycogen_Characterization|Laboratory Notebook]]== |
Latest revision as of 03:49, 19 September 2015