Introduction:

 Inclusion bodies, sometimes called elementary bodies, are nuclear or cytoplasmic aggregates of
stable substances, usually proteins
 PHA: Polyhydroxyalkanoates or PHAs are polyesters produced in nature by numerous
microorganisms, including through bacterial fermentation of sugars or lipids.[1] When produced
by bacteria they serve as both a source of energy and as a carbon store
o The biosynthesis of PHA is usually caused by certain deficiency conditions (e.g. lack of
macro elements such as phosphorus, nitrogen, trace elements, or lack of oxygen) and the
excess supply of carbon sources.
o Polyesters are deposited in the form of highly refractive granules in the cells.
o PHA granules are then recovered by disrupting the cells.
 PHB: Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to the
polyesters class that area of interest as bio-derived and biodegradable plastics. It’s the most
common form of polyhydroxyalkanoates.
 PHA Industrial production:
o Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics
for the past many years. However, PHA still have only a very limited market. Because of
the availability of large amount of shale gas, petroleum will not raise dramatically in price,
this situation makes PHA less competitive compared with low cost petroleum based
plastics. Therefore, two strategies have been adopted to meet this challenge: first, the
development of a super PHA production strain combined with advanced fermentation
processes to produce PHA at a low cost; second, the construction of functional PHA
production strains with technology to control the precise structures of PHA molecules,
this will allow the resulting PHA with high value added applications.
 Halophile: high salt concentrations
 Halomonas campaniensis LS21
o Sea-water based open and continuous process for PHA production
o Isolated from environmental samples collected from China, with the ability of utilizing
mixing substrates including cellulose
o Industrial advantage since it is able to grow in high salt concentrations and high pH, thus
energetic costs for sterilization are reduced
 MreB: cell shape-determining protein, forms filaments, the polymers control/restrict the mobility
of the cell wall elongation enzyme complex
o Plays an important role in maintaining cell shapes by directing the correct insertion of
peptidoglycan precursors, it is also essential for maintaining normal cell growth and cell
rigidity similar to cell walls
o The absence of MreB or MreB-related proteins such as MreC, MreD or RodZ leads to the
loss of rods to spherical shapes
o High concentrations of magnesium (Mg2+) rescue the viability and shape defects of mreBs
and other mutants involved in different aspects of cell wall synthesis by a yet unknown
mechanism
 o It has been proposed that Mg2+ may stiffen the cell wall, compensating for structural
defects associated to the absence of mreB [41]. CM is traditionally supplemented with 5
mM Mg2+
 Since spherical shapes have thelargest ratios of volume to size, we speculated that a change from
rod to sphere may increase PHA accumulation in bacterial cells  Jiang, 2015
 ftZ: plays an important role in the bacterial cell divisionprocess as a tubulin-like protein. Z rings
formed by FtsZ assembly,is very dynamic process
o Many FtsZ inhibitors can directly interact with FtsZ protein and inhibit cell division in
various manners, thus leading tofilamentous cells. Examples of these include SulA, Noc,
SlmA and MinCDE family (Cho et al., 2011)
o SulA overexpression leads to the reduction on the amount of FtsZ
o On the other hand, FtsZ overproduction accelerates cell division, resulting in high cell
density growth

o
 pyrF
o related to uracil production
o can metabolize 5-FOA into a toxic compound
o is a non essential gene in LB medium
o when you grow on mineral medium you will need to have pyrF in the plasmid

Methodology:

 Halomonas double crossover: The first case of gene knock-out in H. bluephagenesis was reported
in 2014
o Upstream and downstream DNA of the target gene (also termed homologous arms) are
joined and cloned into a suicide vector.
o The suicide vector carries a replicon unable to replicate in H. bluephagenesis. Thus, under
the selective pressure of an antibiotic, the suicide vector is integrated into the genome
via homologous recombination. This step is called single crossover.
o Double crossover occurs when stimulated by a helper plasmid. The helper plasmid
harbors the gene of an endonuclease I-SceI. When the endonuclease gene is expressed,
the I-SceI recognizes its corresponding site on the suicide vector (now integrated into the
genome) and creates double-strand break in the genome. Consequently, homologous
recombination happens again to eliminate the double-strand break from the genome,
resulting to either gene knock-out mutant or wild-type strain.
o Finally, the gene knock-out mutant is verified by PCR and sequencing.
  pyrF-based method was developed for efficient gene knock-out in H. campaniensis (Jiang et al.,
2017).
o First, the pyrF gene encoding orotic acid 50-phosphate decarboxylase was deleted from
the chromosome of H. campaniensis.
o Then the I-SceI gene in the above-mentioned suicide vector was replaced with pyrF. After
a single crossover, the pyrF gene together with the whole suicide vector was integrated
into the chromosome. In the presence of 5-fluoroacid acid (5-FOA), PyrF converts 5-FOA
into a toxic metabolite5-fluorouracil.
o Therefore, pyrF can act as a negative selection marker, which stimulates double crossover
to eliminate the pyrF and suicide vector from the chromosome. In H. campaniensis, this
pyrF-based gene knock-out method showed higher efficiency than I-SceI-based method
 A replicon is a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a
single origin of replication.

Results:

Conclusion: