Favorite Parts
- 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] policistronic GlgCAB
- polycistronic methanol assimilation pathway [http://parts.igem.org/Part:BBa_K1585241 BBa_K1585241]
Parts Table
The following table lists all parts we created and submitted to the registry.
BioBrick |
Description |
Submitted to the Registry
|
[http://parts.igem.org/Part:BBa_K1585200 K1585200] |
Mdh2 (Methanoldehydrogenase 2) from Bacillus methanolicus MGA3, codon optimized for E. coli (Basic Part) |
|
[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 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_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_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_K1585300 K1585300] |
GlgA from E. coli |
|
[http://parts.igem.org/Part:BBa_K1585301 K1585301] |
GlgB from E. coli |
|
[http://parts.igem.org/Part:BBa_K1585310 K1585310] |
B0034 with glgA from E. coli |
✓
|
[http://parts.igem.org/Part:BBa_K1585311 K1585311] |
B0034 with glgB from E. coli |
✓
|
[http://parts.igem.org/Part:BBa_K1585312 K1585312] |
B0034 with glgC deltaG336D |
✓
|
[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 (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_K1585350:Design K1585350] |
anti glgX targeting plasmid to generate gene knockout together with pCas |
✓
|
[http://parts.igem.org/Part:BBa_K1585351 K1585351] |
anti glgP targeting plasmid to generate gene knockout together with pCas |
✓
|
[http://parts.igem.org/Part:BBa_K1585100 K1585100] |
Anderson promoter J23100 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585101 K1585101] |
Anderson promoter J23101 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585102 K1585102] |
Anderson promoter J23102 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585103 K1585103] |
Anderson promoter J23103 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585104 K1585104] |
Anderson promoter J23104 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585105 K1585105] |
Anderson promoter J23105 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585106 K1585106] |
Anderson promoter J23106 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585110 K1585110] |
Anderson promoter J23110 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585113 K1585113] |
Anderson promoter J23113 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585115 K1585115] |
Anderson promoter J23115 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585116 K1585116] |
Anderson promoter J23116 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585117 K1585117] |
Anderson promoter J23117 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585118 K1585118] |
Anderson promoter J23118 with lac I binding site |
|
[http://parts.igem.org/Part:BBa_K1585119 K1585119] |
Anderson promoter J23119 with lac I binding site |
|
<groupparts>iGEM015 Aachen</groupparts>
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.
Expression verification & localization of the Mdh
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.
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.
Functionality of the expressed Mdh
To test if the expressed Mdh is functional, we modified a colorimetric and fluorescent formaldehyde assay described by 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 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 20h 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
- 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 37 °C
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 labled methanol experiment along with the BL21 Gold (DE3) expressing GlgC as a negative control, both in multiple replicates (n>35).
The Mdh expressing strain showed significantly more formaldehyde production indicating a functional expression of the Mdh.
K1585211
This is the translational unit of 3-hexulose-6-phosphate synthase (Hps) from Bacillus methanolicus MGA3. It is codon optimized for E. coli.
K1585212
This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from Bacillus methanolicus MGA3. It is codon optimized for E. coli.
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.
K1585310
This is the translational unit of GlgA (Glycogen synthase), codon-optimized for E.coli. The enzyme elongates linear glycogen chains by using ADP-glucose units and forming α-1,4-glycosidic bonds. Through autophosphorylation, GlgA is also the starting point of glycogen synthesis in bacteria.
Usage and Biology
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.
On top of that, the function of the construct was proven by an iodine staining with BL21 Gold (DE3) and BL21 Gold (DE3) expressing glgA in pSB1K30. The iodine staining is performed with Lugol's iodine which dyes glycogen resulting in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the brown color of BL21 Gold (DE3) expressing glgA indicates that the enzyme has the expected activity.
K1585311
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.
Usage and Biology
The construct was confirmed by sequencing. The expression was tested in Bl21 Gold (DE3) strains containing BBa_K1585311 in a [http://parts.igem.org/Part:BBa_K1362093 pSB1K30] expression vector after IPTG induction.
K1585312
This is the translational unit of GlgC, the ADP-glucose pyrophophorylase. It creates ADP-glucose from glucose-1-phosphate for glycogen synthesis.
Usage and Biology
The BioBrick is based on [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] by Team Edinburg 2008, but was fused with the well-characterized RBS B0034.
For expression tests, BBa_K1585312 was cloned into a [http://parts.igem.org/Part:BBa_K1362091 pSB1A30] expression vector and transformed in BL21 Gold (DE3).
On top of that, the function of the construct was proven by an iodine staining with BL21 Gold (DE3) and BL21 Gold (DE3) expressing glgC in pSB1K30. The iodine staining is performed with Lugol's iodine which dyes glycogen resulting in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the brown color of BL21 Gold (DE3) expressing glgC indicates that the enzyme has the expected activity.
K1585320
This composite part combines [http://parts.igem.org/Part:BBa_K1585320| glgA] and [http://parts.igem.org/Part:BBa_K1585320| 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
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].
Usage and Biology
In order to upregulate the whole glycogen synthesis pathway, the polycistronic plasmid was built. The glgC coding sequence is based on the part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016] from Team Edinburgh 2008, but he RBS B0034 was added to the existing Biobrick.
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 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.
K1585241
Polycistronic expression plasmid of Mdh, Hps, Phi and Xpk controlled by Anderson Promoter 19 (J23119), Ribosome binding site B0034 and Terminator B0015
K1585350
This plasmid is targeting glgX in the E. coli genome by expressing a single guide RNA (sgRNA) for CRISPR/Cas9 genome editing. It contains a repair template that disrupts the beginning of the glgX coding sequence and introduces a sequence with multiple stop codons.
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.
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.
K1585351
This plasmid is targeting glgP in the E. coli genome by expressing two sgRNAs for CRISPR/Cas9 genome editing. The complete glgP coding sequence is cut out and multiple stop codons are introduced by the repair templates.
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.
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.
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.