Part:BBa_K1585311
B0034.GlgB
This is the translational unit of GlgB (glycogen branching enzyme) from E. coli. The enzyme catalyzes the formation of alpha-1,6-branches in glycogen synthesis.
Usage and Biology
The construct was confirmed by sequencing. The expression was tested in Bl21 Gold (DE3) strains containing BBa_K1585311 in a pSB1K30 expression vector after IPTG induction.
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| SDS-PAGE of GlgB in comparison to mRFP 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. |
We wanted to investigate the functionality of the strains expressing glgB. Therefore, we used [http://2015.igem.org/Team:Aachen/Notebook/Protocols#Dinitrosalicylic_Acid_Staining dinitrosalicylic acid staining (DNS)] for detection of reducing ends 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.
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).
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| DNS staining of non-hydrolyzed samples of the glgB strain and wild type. 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. |
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| DNS staining of hydrolyzed samples of the glgB strain and wild type The hydrolyzed samples indicate that the glycogen amount is higher in the wild type compared to the glgB 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. |
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.
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| Ratio of non-hydrolyzed and hydrolyzed DNS staining values of the glgB strain and wild type 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. |
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| Glycogen calibration curve With the calibration curve we are able to identify the glycogen concentration of our samples. Error bars show propagation of uncertainty. |
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 465
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