Genetic Engineering and Biotransfomation

1. Biotransformation through Genetic
Engineering
Prepared by:
Parmar SweetyGirishbhai
Guided by:
Dr. Deepesh Bhatt

2. Genetic Engineering

3. Genetic Engineering of Terpenoid
metabolism
• Herbivore damage plants
• Herbivore – Pieris rapae
• Release complex mixtures of volatiles that
attract enemies of herbivore.
• Volatiles – Terpenoid
• It attract carnivore
• wasp Cotesia rubecula

4. Schematicrepresentationof biosynthetic
pathway
CoxIV-FaNES1 construct scheme
4,8,12-trimethyl- 1,3(E),7(E),11-
tridecatetraene E(E,E)-TMTT)

5. (A)Undamaged wild type;
(B)wild type, infested by spider mites
for 10 days;
(C)wild type, infested by P. rapae for
24 hours;
(D) undamaged CoxIV-FaNES1;
(E)undamaged CoxIVFaNES1;
(F)wild type, detached leaves placed
with petiole in (E)-nerolidol (10
mg/ml) for 4 hours before
headspace sampling;
(G)CoxIVFaNES1, plant 2.2 sprayed
with 5 mM jasmonic acid 24 hours
before headspace sampling.
(H)Identified compounds: (a) (E)-
DMNT; (b) (3S)-(E)-nerolidol; (c)
(E)-TMTT.

6. Genome Editing

7. Genome Editing via ZFN

8. Deveolpment of Zinc finger

11. Disadvantages of ZFN
• Laborious,
• Resource intensive,
• Time consuming,
• Expensive
• They suffer from high failure rates

12. Transcription Activator Like Effector
Nucleases
• Xanthomonas
• Type III secretion system
• Highly conserved 33–34 amino acid sequence
with divergent 12th and 13th amino acids.
• These two positions, referred to as the Repeat
Variable Diresidue (RVD)
• Fokl - Flavobacterium okeanokoites

13. TALENs

14. Modifications in TALENs
• The unique biochemical properties of the I-
TevI nuclease make it usable instead of fokl.
• It is expected that the mTALENs-TevI, that is,
mTALENTs will be able to cleave DNA in a
site-specific fashion.
• Significantly lower the potential off-target
events

15. Sickle cell anemia

16. Correction of the Sickle Cell Disease

17. References
1. Kappers, I. F., Aharoni, A., Van Herpen, T. W., Luckerhoff, L.
L., Dicke, M., & Bouwmeester, H. J. (2005). Genetic
engineering of terpenoid metabolism attracts bodyguards to
Arabidopsis. Science, 309(5743), 2070-2072.
2. Mahfouz, M. M., Piatek, A., & Stewart, C. N. (2014).
Genome engineering via TALENs and CRISPR/Cas9
systems: challenges and perspectives. Plant biotechnology
journal, 12(8), 1006-1014.
3. Sun, N., & Zhao, H. (2014). Seamless correction of the sickle
cell disease mutation of the HBB gene in human induced
pluripotent stem cells using TALENs. Biotechnology and
bioengineering, 111(5), 1048-1053