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] | ||
=Parts Table= | =Parts Table= | ||
− | The following table lists all parts we created and | + | The following table lists all parts we created and submitted to the registry. |
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! BioBrick !! Description !! Submitted to the Registry | ! BioBrick !! Description !! Submitted to the Registry | ||
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585200 K1585200] || Mdh2 (Methanoldehydrogenase 2) from ''Bacillus methanolicus | + | |[http://parts.igem.org/Part:BBa_K1585200 K1585200] || Mdh2 (Methanoldehydrogenase 2) from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' ([[Team:Aachen/Basic_Part|Basic Part]]) || |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585201 K1585201] || Hps (3-hexulose-6-phosphate synthase) from ''Bacillus methanolicus | + | |[http://parts.igem.org/Part:BBa_K1585201 K1585201] || Hps (3-hexulose-6-phosphate synthase) from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' || |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585202 K1585202] || Phi (6-Phospho-3-hexuloisomerase) from ''Bacillus methanolicus | + | |[http://parts.igem.org/Part:BBa_K1585202 K1585202] || Phi (6-Phospho-3-hexuloisomerase) from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' || |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585203 K1585203] || Xpk (D-Xylulose-5-phosphate-phosphoketolase) from ''Bifidobacterium adolescentis'', codon optimized for ''E.coli'' || | + | |[http://parts.igem.org/Part:BBa_K1585203 K1585203] || Xpk (D-Xylulose-5-phosphate-phosphoketolase) from ''Bifidobacterium adolescentis'', codon optimized for ''E. coli'' || |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585210 K1585210] || B0034 with Mdh2 from ''Bacillus methanolicus'' MGA3, codon optimized for ''E.coli'' || ✓ | + | |[http://parts.igem.org/Part:BBa_K1585210 K1585210] || B0034 with Mdh2 from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' || ✓ |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585211 K1585211] || B0034 with Hps from ''Bacillus methanolicus'' MGA3, codon optimized for ''E.coli'' || ✓ | + | |[http://parts.igem.org/Part:BBa_K1585211 K1585211] || B0034 with Hps from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' || ✓ |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585212 K1585212] || B0034 with Phi from ''Bacillus methanolicus'' MGA3, codon optimized for ''E.coli'' || ✓ | + | |[http://parts.igem.org/Part:BBa_K1585212 K1585212] || B0034 with Phi from ''Bacillus methanolicus'' MGA3, codon optimized for ''E. coli'' || ✓ |
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585213 K1585213] || B0034 with Xpk from ''Bifidobacterium adolescentis'', codon optimized for ''E.coli'' || ✓ | + | |[http://parts.igem.org/Part:BBa_K1585213 K1585213] || B0034 with Xpk from ''Bifidobacterium adolescentis'', codon optimized for ''E. coli'' || ✓ |
|- | |- | ||
|[http://parts.igem.org/Part:BBa_K1585300 K1585300] || GlgA from ''E. coli'' || | |[http://parts.igem.org/Part:BBa_K1585300 K1585300] || GlgA from ''E. coli'' || | ||
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|[http://parts.igem.org/Part:BBa_K1585320 K1585320] || Combined translational unit of of glgA and glgB || ✓ | |[http://parts.igem.org/Part:BBa_K1585320 K1585320] || Combined translational unit of of glgA and glgB || ✓ | ||
|- | |- | ||
− | |[http://parts.igem.org/Part:BBa_K1585321 K1585321] || Combined translational unit of glgC, glgA and glgB | + | |[http://parts.igem.org/Part:BBa_K1585321 K1585321] || Combined translational unit of glgC, glgA and glgB ([[Team:Aachen/Composite_Part|Composite Part]]) || ✓ |
|- | |- | ||
|[http://parts.igem.org/Part:BBa_K1585241 K1585241] || Polycistronic expression plasmid of Mdh, Hps, Phi and Xpk controlled by Anderson Promoter 19 (J23119), Ribosome binding site B0034 and Terminator B0015 || ✓ | |[http://parts.igem.org/Part:BBa_K1585241 K1585241] || Polycistronic expression plasmid of Mdh, Hps, Phi and Xpk controlled by Anderson Promoter 19 (J23119), Ribosome binding site B0034 and Terminator B0015 || ✓ | ||
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|} | |} | ||
− | + | <groupparts>iGEM015 Aachen</groupparts> | |
==K1585210== | ==K1585210== | ||
− | This is the translational unit of methanol dehydrogenase 2 from Bacillus methanolicus MGA3. It is codon optimized for E.coli. The mdh catalyzes the conversion of methanol to formaldehyde. | + | This is the translational unit of methanol dehydrogenase 2 from ''Bacillus methanolicus'' MGA3. It is codon optimized for ''E. coli''. The mdh catalyzes the conversion of methanol to formaldehyde. |
<center> | <center> | ||
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'''Expression verification & localization of the Mdh''' | '''Expression verification & localization of the Mdh''' | ||
− | The expression of the Mdh behind a T7 promoter was verified by doing | + | The expression of the Mdh behind a T7 promoter was verified by doing a SDS-PAGE. To localize the Mdh not only whole cells were used but also the fragments of lysed cells and the respective supernatant. The expected bands were clearly visible in all samples proving the expression of the Mdh. However, the Mdh specific band had the highest intensity in the sample of the cell fragments whereas the same band was only of slight intensity in the supernatant. This indicates that the Mdh is incorporated into inclusion bodies. |
− | {{Team:Aachen/Figure|Aachen 15-09-03 MDH Assay expression check + new constitutive MDH + Poly.png|title= Expression test of #IGEM# |subtitle= | + | {{Team:Aachen/Figure|Aachen 15-09-03 MDH Assay expression check + new constitutive MDH + Poly.png|title= Expression test of #IGEM# |subtitle= A SDS-PAGE of the whole cell, the cell fragments and the lysate supernatant of the Mdh expressing BL21 Gold (#IGEM#) and the GlgC expressing BL21 Gold (#OHES#) as a negative control was run. The expected band at the weigth of the Mdh can be clearly seen in all #IGEM# samples. The according band with the strongest intensity occurs in the lane with #IGEM# fragments indicating the formation of inclusion bodies containg the Mdh. |size=medium}} |
− | To avoid the incorporation of the Mdh into inclusion bodies, the alternative ''E. coli'' strains SHuffle T7 Express and C43 were used. SHuffle T7 Express allows more efficient protein folding in the cytoplasm and lacks proteases whereas C43 allows the expression of toxic proteins. Additionally, cultivation at a lower temperature of | + | To avoid the incorporation of the Mdh into inclusion bodies, the alternative ''E. coli'' strains SHuffle T7 Express and C43 were used. SHuffle T7 Express allows more efficient protein folding in the cytoplasm and lacks proteases whereas C43 allows the expression of toxic proteins. Additionally, cultivation at a lower temperature of 30 °C was tested. It was shown that all strains were able to grow on M9 medium, however, another test for the expression and localization of the Mdh did not reveal major differences between the strains. In every case the Mdh was still incorporated into inclusion bodies. |
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To test if the expressed Mdh is functional, we modified a colorimetric and fluorescent formaldehyde assay described by [[2015.igem.org/Team:Aachen/Notebook/Protocols#Nash Assay#|T. Nash]]. In the in the presence of formaldehyde the yellow and fluorescing diacetyl-dihydro lutidine is formed. In the reaction catalyzed by the Mdh methanol is converted to formaldehyde which can be detected by the mentioned assay. | To test if the expressed Mdh is functional, we modified a colorimetric and fluorescent formaldehyde assay described by [[2015.igem.org/Team:Aachen/Notebook/Protocols#Nash Assay#|T. Nash]]. In the in the presence of formaldehyde the yellow and fluorescing diacetyl-dihydro lutidine is formed. In the reaction catalyzed by the Mdh methanol is converted to formaldehyde which can be detected by the mentioned assay. | ||
− | First, the different cells as well as their respective fragments and lysate supernatants were screened for formaldehyde production. The samples were taken | + | 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 | + | * More formaldehyde was produced in the samples taken 6 h after induction |
− | * Strains cultivated at | + | * Strains cultivated at 37 °C show a stronger response than the same ones cultivated at 30 °C |
− | * The assay works better and faster at an incubation temperature of | + | * The assay works better and faster at an incubation temperature of 37 °C |
{{Team:Aachen/Figure|Aachen_Comparison_strains_@_different_conditions.png|title=Comparison of different strains at varying cultivation conditions. |subtitle=The highest activity could be shown in BL21 Gold (DE3) at a cultivation temperature of 37 °C in M9 in a sample taken 6 h after induction.|size=medium}} | {{Team:Aachen/Figure|Aachen_Comparison_strains_@_different_conditions.png|title=Comparison of different strains at varying cultivation conditions. |subtitle=The highest activity could be shown in BL21 Gold (DE3) at a cultivation temperature of 37 °C in M9 in a sample taken 6 h after induction.|size=medium}} | ||
− | By far the highest formaldehyde production was observed in the BL21 Gold (DE3) cells | + | By far the highest formaldehyde production was observed in the BL21 Gold (DE3) cells 6 h after induction cultivated at 37 °C despite its formation of inclusion bodies. |
To show the significance of the production of formaldehyde the assay was conducted with the Mdh expressing BL21 Gold (DE3) cultivated in the [[2015.igem.org/Team:Aachen/Lab/Methanol/Labeling_Experiment|labled methanol experiment]] along with the BL21 Gold (DE3) expressing GlgC as a negative control, both in multiple replicates (n>35). | To show the significance of the production of formaldehyde the assay was conducted with the Mdh expressing BL21 Gold (DE3) cultivated in the [[2015.igem.org/Team:Aachen/Lab/Methanol/Labeling_Experiment|labled methanol experiment]] along with the BL21 Gold (DE3) expressing GlgC as a negative control, both in multiple replicates (n>35). | ||
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==K1585211== | ==K1585211== | ||
− | This is the translational unit of 3-hexulose-6-phosphate synthase (Hps) from Bacillus methanolicus MGA3. It is codon optimized for E.coli. | + | This is the translational unit of 3-hexulose-6-phosphate synthase (Hps) from ''Bacillus methanolicus'' MGA3. It is codon optimized for ''E. coli''. |
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==K1585212== | ==K1585212== | ||
− | This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from Bacillus methanolicus MGA3. It is codon optimized for E.coli. | + | This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from ''Bacillus methanolicus'' MGA3. It is codon optimized for ''E. coli''. |
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==K1585213== | ==K1585213== | ||
− | This is the translational unit for the D-Xylulose-5-phosphate-phosphoketolase (Xpk). It derives from Bifidobacterium adolescentis and is codon optimized for E. coli. | + | This is the translational unit for the D-Xylulose-5-phosphate-phosphoketolase (Xpk). It derives from Bifidobacterium adolescentis and is codon optimized for ''E. coli''. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | The construct was confirmed by sequencing. Moreover, the expression of ''glgA'' was tested by | + | The construct was confirmed by sequencing. Moreover, the expression of ''glgA'' was tested by a SDS-PAGE. In the picture below, you can see that ''glgA'' is expressed in strains containing BBa_K1585310 in a [http://parts.igem.org/Part:BBa_K1362093 pSB1K30] expression vector. |
{{Team:Aachen/Figure|Aachen_15-07-03_glgA_expression_test_wiki.png|title=SDS-PAGE of GlgA in comparison to mRFP|subtitle=After induction with IPTG at OD=0.6, ''glgA'' was expressed. Samples were taken 6 and 18.5 hours after induction. The small arrows indicates the expected bands for GlgA. mRFP in pSB1K30 (T7 promoter) was used as the negative control.|size=medium}} | {{Team:Aachen/Figure|Aachen_15-07-03_glgA_expression_test_wiki.png|title=SDS-PAGE of GlgA in comparison to mRFP|subtitle=After induction with IPTG at OD=0.6, ''glgA'' was expressed. Samples were taken 6 and 18.5 hours after induction. The small arrows indicates the expected bands for GlgA. mRFP in pSB1K30 (T7 promoter) was used as the negative control.|size=medium}} | ||
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==K1585311== | ==K1585311== | ||
− | This is the translational unit of GlgB (glycogen branching enzyme) from E. coli. The enzyme catalyzes the formation of | + | This is the translational unit of GlgB (glycogen branching enzyme) from ''E. coli''. The enzyme catalyzes the formation of α-1,6-branches in glycogen synthesis. |
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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== | ||
− | This composite part combines all 3 enzymes involved in glycogen synthesis. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates | + | This composite part combines all 3 enzymes involved in glycogen synthesis. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. This construct is an extension and improvement of [http://parts.igem.org/Part:BBa_K118016 BBa_K118016]. |
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The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in Bl21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector. | The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in Bl21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector. | ||
− | {{Team:Aachen/Figure|Aachen_glgCAB_for_registry.png|title=SDS-PAGE of ''glgCAB'' in pSB1A30|subtitle=glgCAB in pSB1A30 was expressed in Bl21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes . The Bl21 Gold (DE3) wild type was used as negative control.|size=medium}} | + | {{Team:Aachen/Figure|Aachen_glgCAB_for_registry.png|title=SDS-PAGE of ''glgCAB'' in pSB1A30|subtitle=glgCAB in pSB1A30 was expressed in Bl21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes. The Bl21 Gold (DE3) wild type was used as negative control.|size=medium}} |
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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|Aachen_glgCAB_, | + | {{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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===Usage and Biology=== | ===Usage and Biology=== | ||
− | The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the ''glgX'' coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the ''glgX'', you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in E. coli, it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT. | + | The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the ''glgX'' coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the ''glgX'', you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in ''E. coli'', it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT. |
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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===Usage and Biology=== | ===Usage and Biology=== | ||
− | The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the ''glgP'' coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the ''glgP'', you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in E. coli, it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT. | + | The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the ''glgP'' coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the ''glgP'', you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in ''E. coli'', it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT. |
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. | ||
Sequencings of the respective genomic region have shown that ''glgP'' was successfully cut out. | Sequencings of the respective genomic region have shown that ''glgP'' was successfully cut out. | ||
− | In further experiments, the effect of this gene knockout was characterized. The expected consequence was an accumulation of glycogen due to the absense of a glycogen phosphorylase degrading | + | In further experiments, the effect of this gene knockout was characterized. The expected consequence was an accumulation of glycogen due to the absense of a glycogen phosphorylase degrading α-1,4-linked glucose. We confirmed this by iodine staining with Lugol's iodine, which dyes glycogen in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the BL21 Gold (DE3) ∆''glgP'' strain is much darker than the wild type. |
{{Team:Aachen/Figure|Aachen_15-09-05_M9_WT,_delta_P.png|title=''glgP'' gene knockout|subtitle=The knockout was achieved cutting out most of the coding sequence of ''glgP'', thereby inserting a short coding sequence that contains multiple stop codons in both frames.|size=medium}} | {{Team:Aachen/Figure|Aachen_15-09-05_M9_WT,_delta_P.png|title=''glgP'' gene knockout|subtitle=The knockout was achieved cutting out most of the coding sequence of ''glgP'', thereby inserting a short coding sequence that contains multiple stop codons in both frames.|size=medium}} | ||
{{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