Composite

Part:BBa_K3385022

Designed by: Daniel Bavnhøj   Group: iGEM20_DTU-Denmark   (2020-10-14)


CRISPR_gul-1_KO

Theoretical expectation: gul-1 was found in literature about the fungi Neurospora crassa where it encodes an mRNA binding protein involved in remodeling of the cell wall. A homolog was found in A. niger. The expected morphology of the Δgul-1 mutant was hyperbranched pellets.

Full guide construct for knockout of the uncharacterized N. crassa ortholog gul-1 gene (AN08g03530) in A. niger. The BioBlocks can be USER cloned into the PacI/Nt.BbvCI digested pFC330 backbone.


Plasmid map of pFC330[1].


Functionality: The sgRNA efficiency has been accessed through the technique to assess protospacer efficiency (TAPE) [2]. A repair oligo is used to mediate homologous recombination, where a highly efficient sgRNA will show no colonies without the repair oligo, while less efficient sgRNA will show a reduced number of colonies.

Results: Below is a picture showing A. niger transformed with CRISPR_gul-1_KO and the repair oligo for gul-1. It shows efficient gene deletion when it's transformed with a repair oligo.
TAPE showing sgRNA efficiency.


To see if the K/O’s were successful, other than looking at macromorphology, tissue PCRs were performed. By the amplification of specific primers, upstream and downstream of the gene, it can be verified if the gene has successfully been knocked out. If it has been knocked out the primers are gonna be closer to each other resulting in a smaller band in the Tissue PCR. However if the gene is still present in the genome, the band size will be the same as the target gene as seen in the table below.



Expected length of each K/O
Targeted gene Expected gene length after K/O Control lenght
ΔspaA 672 bp 3528 bp
Δgul-1 545 bp 5022 bp
ΔpkaR 370 bp 1661 bp
Picture of the tissue PCRs performed on ΔspaA, Δgul-1 and ΔpkaR.
Summary: Protein secretion in the strain was highly increased and had a slightly lower growth rate in the bioreactor when compared to the reference. It presents an almost 2-fold increase in branching frequency, making this mutant a very good candidate for an improved cell factory for protein production.

Radar chart showing 6 different parameters of Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the summary section on the result page.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).


Microscope pictures and Simulation model

Left: Confocal microscope picture of Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.


Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of Δgul-1 growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating Δgul-1:
  • Branching frequency: 0.0300301
  • Gamma distribution parameters used for curvature angles: (0.6220764, 8.0102895)
  • Beta distribution parameters used for branching angles: (3.2414641, 0.8869618)
  • Experimental growth rate: 0,314625


BioLector

Comparing the growth kinetics of the Δgul-1 mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase followed by a slightly shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μMax0.32h-1 versus μMax0.28h-1.

Growth profile of Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the Δgul-1 mutant in bioreactors, it showed a lag phase equivalent to that of the reference strain ATCC 1015 followed by a longer exponential growth phase. Δgul-1 has a slightly lower growth rate than the reference strain, at μMax0.31h-1 and μMax0.32h-1 versus μMax0.37h-1 and μMax0.33h-1. Glucose levels start decreasing two thirds of the way through growth and keep decreasing for over 10 hours after the strain enters the stationary phase.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by Δgul-1 during the fermentation in g/L (dark green).



Light microscope picture of Δgul-1 after 23 hours of fermentation with a 20X magnification.



The microscope picture from the bioreactor sample for Δgul-1 has a similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched phenotype in a more dispersed network.

Protein secretion

Glucoamylase activity has a significant 2-fold increase in this mutant compared with the reference strain. Such high glucoamylase activities were reach that the total activity was not measured. From the flat curve it could be expected that the glucoamylase activity would have reached even higher levels have it been measured.

Glucoamylase activity in UA/mL of Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.


The total protein secretion was to the reference strain and thereby the specific activity was substantially higher. This might be due to a different protein secretion profile that has a similar amount of protein being secreted but where the family of glucoamylases are preferentially produced.

Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 489
    Illegal EcoRI site found at 660
    Illegal EcoRI site found at 831
    Illegal SpeI site found at 978
    Illegal PstI site found at 544
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 489
    Illegal EcoRI site found at 660
    Illegal EcoRI site found at 831
    Illegal NheI site found at 333
    Illegal SpeI site found at 978
    Illegal PstI site found at 544
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 489
    Illegal EcoRI site found at 660
    Illegal EcoRI site found at 831
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 489
    Illegal EcoRI site found at 660
    Illegal EcoRI site found at 831
    Illegal SpeI site found at 978
    Illegal PstI site found at 544
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 489
    Illegal EcoRI site found at 660
    Illegal EcoRI site found at 831
    Illegal SpeI site found at 978
    Illegal PstI site found at 544
    Illegal AgeI site found at 204
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 162


References:
[1] A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. Nodvig CS, Nielsen JB, Kogle ME, Mortensen UH. PLoS One. 2015 Jul 15;10(7):e0133085. doi: 10.1371/journal.pone.0133085. eCollection 2015. PONE-D-15-11561 [pii] PubMed 26177455

[2] Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli. Nodvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, Mortensen UH. Fungal Genet Biol. 2018 Jan 8. pii: S1087-1845(18)30004-5. doi: 10.1016/j.fgb.2018.01.004. 10.1016/j.fgb.2018.01.004 PubMed 29325827