Composite

Part:BBa_K3385018

Designed by: Daniel Bavnhøj   Group: iGEM20_DTU-Denmark   (2020-10-13)


CRISPR_spaA_KO

Theoretical expectation: It has been suggested that spaA encodes a molecule which is involved in polarity maintenance and thereby polar growth. The mutant was expected to have a dense hyperbranched morphology.

Full guide construct for knockout of the Polarisome component gene (spaA) in A. niger. This BioBlock has to be 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_spaA_KO and the repair oligo for spaA. 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: The strain presents a shorter lag phase than the reference strain and the highest growth rate in the BioLector, although this is not seen in the bioreactor. It does not have a great increase in hyperbranching and protein secretion is very similar to the reference strain.

Radar chart showing 6 different parameters of ΔspaA 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 ΔspaA at 10X magnification af 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 ΔspaA growing for 12 hours (using experimental growth rate from ou BioLector data) is seen above.
Parameters specific for simulating ΔspaA:
  • Branching frequency: 0.0182251
  • Gamma distribution parameters used for curvature angles: (1.6948611, 2.8805343)
  • Beta distribution parameters used for branching angles: (5.1349854, 1.2365410)
  • Experimental growth rate: 0.345225


BioLector

Comparing the growth kinetics of the ΔspaA mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase and a shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μMax0.35h-1 versus μMax0.28h-1.

Growth profile of ΔspaA 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 ΔspaA mutant in bioreactors, it showed a short lag phase followed by a long exponential growth phase compared with the reference strain ATCC 1015. The ΔspaA mutant has a lower growth rate than the reference, at μMax0.19h-1 and μMax0.20h-1 versus μMax0.42h-1 for both duplicates. The fermentation was accidentally run with overpressure and is therefore only directly comparable with the ΔchsC mutant strain and with one run of the ATCC 1015 fermentations, due to these bioreactors also being run with overpressure. Glucose levels decrease slowly in the beginning and then decrease fast after the mutant enters stationary phase.

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



Light microscope picture of ΔspaA after 22 hours of fermentation with a 20X magnification.



The microscope picture from the bioreactor sample for ΔspaA has a similar morphology to that of the brightfield microscopy pictures seen above.

Protein secretion

Glucoamylase activity for the mutant strain shows a similar tendency to the reference strain, with one of the duplicates having lower final value.

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


Protein secretion is not consistent but when computing the specific activity per mg of protein, it can be seen that the mutant again presents similar values to the reference strain.

ΔspaA 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
  • 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
  • 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
  • 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 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