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 entered into the registry.

<--<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.

Catalysed reaction by Mdh Methanol + NAD → Formaldehyde + NADH + H+.

Expression verification & localization of the Mdh

The expression of the Mdh behind a T7 promoter was verified by doing an 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.

Expression test of #IGEM# An 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.

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 6h 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 6h 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

Comparison of different strains at varying cultivation conditions. 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.

By far the highest formaldehyde production was observed in the BL21 Gold (DE3) cells 6h 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).

Formaldehyde formation during in vivo assay #IGEM# expresses Mdh and #OHES# expresses glgC as a negative control (4.5 h after induction). Both constructs were incorporated in vector pSB1A30 and the strains were cultivated at 37 °C. Each value is made from at least 35 replicates of each construct.

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.

Catalysed reaction by Hps Formaldehyde + Ribulose-5-phosphat → D-arabino-hex-3-ulose 6-phosphate.

K1585212

This is the translational unit of the 6-phospho-3-hexuloisomerase (PHI) from Bacillus methanolicus MGA3. It is codon optimized for E.coli.

Catalysed reaction by Phi D-arabino-hex-3-ulose 6-phosphate → D-fructose 6-phosphate.

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.

Catalysed reaction by Xpk D-Xylulose-5-phosphate + phosphate → Acetyl-phosphate + D-Glyceraldehyde-3-phosphate.

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 an 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.

SDS-PAGE of GlgA in comparison to mRFP 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.

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.

Iodine staining of BL21 Gold (DE3) with glgA in pSB1K30 compared to BL21 Gold (DE3) wild type Iodine staining was performed with overnight cultures which were adjusted to the same OD. The darker color of the strain expressing glgA shows that more glycogen is present. Therefore, the functionality of the glycogen synthase is shown.

K1585311

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 [http://parts.igem.org/Part:BBa_K1362093 pSB1K30] expression vector after IPTG induction.

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.

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).

SDS-PAGE of GlgC in comparison to mRFP glgC was expressed in pSB1A30 and compared to mRFP in pSB1A30 (T7 promoter) expression. After induction with IPTG at OD=0.6, glgC was expressed, indicated by the small arrows pointing to the GlgC bands of 48 kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were no longer present.

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.

Iodine staining of BL21 Gold (DE3) with glgC in pSB1K30 compared to BL21 Gold (DE3) wild type Iodine staining was performed with LB and LB + 40 mM glucose overnight cultures which were adjusted to the same OD. The cultures grown in medium with extra glucose were darker than those grown on just LB medium. The darker color of the strain expressing glgC in both samples shows that more glycogen was present. Therefore, the functionality of the ADP-glucose pyrophosphorylase is proved.

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 (alpha-1,4 linked) whereas GlgB uses existing chains to form alpha-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 alpha-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of alpha-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.

SDS-PAGE of glgCAB in pSB1A30 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.

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 alpha-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.

glgP gene knockout 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.

Iodine staining of BL21 Gold (DE3) glgP knockout compared to BL21 Gold (DE3) wild type 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.