iGEM 2018 Marbug

iGEM 2018 Marbug Vibrio_natriegens

Vibrio natriegens

Why V.natriegens can be a candidate genetic chassis?

• it has a very similar metabolism to the well-studied E. coli.
• The specific uptake of many carbon sources per gram dry weight per time was shown to be significantly higher.
• Many commercially interesting compounds are already produced by unmodified V. natriegens, such as alanine.
• pH in the medium can be regulated by aeration.
• The dependence on Na+ ions for its metabolic activity conveys a form of natural biocontainmen (Biosafety), and there is continued record of research since 1958 with not a single documented incident of a human infection with V. natriegens.
• It has the highest rate of transformation in competent species induced be IPTG.

Why is V.natriegens superior to E.coli?

faster!

Doubleing rate:

9.8 minutes at first, and 7 minutes in their own data.

Why?

• Two chromosome
• more rRNA

Potential harmness:

V.natriegens is pathogenic in several marine crustacea, most notably in the swimming crab Portunus trituberculatus, which is farmed commercially in aquaculture in south-east China.

Strain used:

ATCC 14048

The modification needs to be done on wild V.natriegens:

• It can be stored at -80 celcius with 20% glycerol indefinitely.

Multiplex Genome Editing by Natural Transformation (MuGENT) can be used to rapidly generate V.natriegens strains with several large, scarless genome edits without off- targeting effects at high efficiency.

Chromosomal Integration (FLP/FRT system):

Use homogeneous recombination of C+ gene and 1kb homology flanks, from the different region of the genome, using Q5 fusion PCR.

When used with FLP/FRT system:

To establish FLP/FRT system:

First, to introduce the linear fragment into the genome by natural transforming with the help of tfox expression, because of its high efficency.

Use the illustration above

To use the strain after completion of the modifications it is important to get the plasmid out.

With his proposal to grow a culture with the add of IPTG and than to screen for clones who are no longer carbenicillin resistance, we are able to get the pMMB-tfox Plasmid out of the strains.

Then, to introduce a functional FLP recombinase into the cells.

pBR-FLP plasmid

Therefore, it adds a single FRT site into the genome, ready for FLP/FRT insertion.

To choose integration site, they use MATLAB program to select the location which stands between CDSs has has a length more than 500 bp.

This resulted in 24 integration sites, of which 7 were not suitable because they had essential tRNA coding regions which were not detected by the Mathlab program.

By measuring the β-galactosidase’s activity it is possible to draw conclusions about the transcription level at the different integration sites.
-> 12, 15, 19, 20, 22

Engineering

Therefore, they use this technique to constuct three types of V.natriegens:

• VibriClone
• VibriExpress
• VibriInteract

VibriClone:

• Exonuclease ExeM and Dns knock out
• RecA point mutation, 158 Gly->Asp
• KatG knock in
• Lactose operon (LacI, LacZΩ, LacY, LacA) knock in with native partial LacA knock out
• Future work: deleting methylase and restriction modification systems.

VibriExpress:

• T7 RNA polymerase under the control of lacUV5 with LacI gene knock in
• Lon protease knock out
• No OmpT like protease in V.natriegens

Two different strategies to create the T7 integration:

VibriInteract:

Protein interaction studies can be done by several methods:

• Biochemical Methods
  e.g. Co-immunoprecipitation
• Biophysical and Theoretical Methods
  e.g. Fluorescence Resonance Energy Transfer (FRET)
• Genetic Methods
  e.g. Two-Hybrid Systems

Vibrio Two-Hybrid (V2H):

• BA894_1512, similar to Cya gene in E.coli, knock out
• BA894_04835, similar to hsdR gene in E.coli, knock out

Again, why is V.natriegens superior to E.coli:

• Faster
• Natural competence
• Yielding more soluble protein
• Less inclusion bodies

Appication:

Production of 3-Hydroxypropionic acid 3HPA

Need to transfer two enzymes:

·

Sensor-based product screening

1. For 3HPA screening:
   
2. For malonyl-CoA screeming:
   

Direct evolution:
Use MAGE with FACS to generate a library and select the phenotype to minic the process of evolution.

Additionally

Culture:

• 3 mL culture in such a modified 50 mL tube should provide the cells with the maximum possible oxygen, additionally increasing the growth.

Golden-gate-based cloning toolbox

Construction of novel part entry vector

LVL0 entry vector:
----Ori-C+-BsmBI-BsaI-GFP/sfGFP-BsaI-BsmBI----

LVL0 plasmid:
----Ori-C+-BsmBI-BsaI-LVL0 part-BsaI-BsmBI----

LVL0 entry resistance vector:
----Ori-GFP/sfGFP-BsmBI-BsaI-C+-BsaI-BsmBI----

LVL0 resistance plasmid:
----Ori-GFP/sfGFP-BsmBI-BsaI-K+-BsaI-BsmBI----

To form a LVL1 plasmid:
-LVL0 part1--LVL0 part2--LVL0 part3--LVL0 K+-

K+, NO fluorescence

Choice of fusion sites

Principles to obtain optimal fusion sites:

• The newly designed fusion sites must neither be identical to already existing fusion sites nor be palindromic to prevent assembly in a wrong order.
• The fusion sites should not consist of bases that represent a portion of the recognition sequence of a restriction enzyme.
• The remaining candidates were sorted according to their GC content and the fusion sites with the highest GC content were chosen.

To prevent mistranslated proteins:

Two kinds of connectors:

• short connectors
• long connectors (insulators)

The quality of the insulator is characterised by four standard:

• secondary structure
• repeats
• forbidden motifs
• homology to the genome