Part:BBa_K4077003
Modified Pichia Pastoris Plasmid (pPIC9k)
Modified Pichia Pastoris plasmid with Melanocarpus albomyces laccase gene inserted.
partinfo>BBa_K4077003</partinfo>
The pPIC9K vector is identical to pPIC9 except for the presence of the kanamycin resistance gene for in vivo screening of multiple copy inserts. pPIC9K is functional in Pichia strains GS115 and KM71.
Additional features of the pPIC9K vector include:
• 9276 bp fusion vector
• Four unique restriction sites for cloning in frame with the α-factor secretion signal: SnaB I, EcoR I, Avr II, Not I
• Secreted expression of your gene using the α-factor secretion signal
• For expression, your gene must be cloned in frame with the initiation codon of the signal sequence.
• HIS4 selection in Pichia
• For gene replacement at AOX1 in GS115, linearize with Bgl II (generates His+ MutS)
• For insertion at AOX1 in GS115 or KM71, linearize with Sac I (generates His+ Mut+ in GS115 and His+ MutS in KM71)
• For insertion at HIS4, linearize with Sal I (generates His+ Mut+ in GS115 and His+ MutS in KM71)
This modified Pichia pastoris Plasmid (pPIC9k) has a Melanocarpus albomyces laccase gene inserted that expresses the laccase enzyme that was selected as our ideal enzyme through dry lab experiments. This enzyme has a tendency to degrade Gossypol that is toxic present in Cottonseed which is the main aim of our project.
We have used this plasmid in our project to express our enzyme of interest ie. Melanocarpus albomyces laccase. Here are some results of Cloning and expression of laccase gene in P. pastoris GS115.
The 1.6 Kb codon optimized laccase gene of Melanocarpus albomyces was cloned downstream of signal peptide α- mating factor of pPIC9K vector for extracellular protein expression. The resultant recombinant vector was transformed into P. pastoris GS115, and the recombinant strain is designated as GS-COL. The recombinant strain is evaluated for the production of laccase in the shake flask.
Shake flask studies for laccase expression
The recombinant strain GS115-COL was pre-cultured in BMGY medium, where Pure glycerol and crude glycerol, in both mediums the recombinant strain showed excellent growth displaying its ability to consume the impurities associated with crude glycerol. Further the pre-inoculum was transferred to BMMH medium and induced with 1% methanol.
The maximum production of laccase was found to be 9.75 U/ml at 48 h time interval with concomitant increase in cell concentration of 8.9 using pure glycerol based pre-inoculum (Fig 1A). Similarly using crude glycerol based pre inoculum the maximum laccase and cell concentration was found to be 6.75 and 7.59, respectively (Fig 1B). The laccase is clearly displayed a growth associated product. The crude extract will be used for the treating CSM.
References
(User Manual PPIC9K a Pichia Vector for Multicopy Integration and Secreted Expression Catalog No. V175-20)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1222
Illegal XbaI site found at 3703
Illegal PstI site found at 971
Illegal PstI site found at 6295
Illegal PstI site found at 7536
Illegal PstI site found at 10091 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1222
Illegal NheI site found at 3464
Illegal NheI site found at 4411
Illegal PstI site found at 971
Illegal PstI site found at 6295
Illegal PstI site found at 7536
Illegal PstI site found at 10091
Illegal NotI site found at 2905 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1222
Illegal BglII site found at 1
Illegal BglII site found at 2257
Illegal BglII site found at 2560
Illegal BglII site found at 8545
Illegal BamHI site found at 938
Illegal XhoI site found at 1192
Illegal XhoI site found at 7380 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1222
Illegal XbaI site found at 3703
Illegal PstI site found at 971
Illegal PstI site found at 6295
Illegal PstI site found at 7536
Illegal PstI site found at 10091 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1222
Illegal XbaI site found at 3703
Illegal PstI site found at 971
Illegal PstI site found at 6295
Illegal PstI site found at 7536
Illegal PstI site found at 10091
Illegal AgeI site found at 2971 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5751
Illegal BsaI site found at 7852
Illegal BsaI.rc site found at 2286
Illegal BsaI.rc site found at 9917
Illegal SapI site found at 8834
| None |