We use pGAPZ A vector from invitrogen and insert this part to construct our vector. We constructed this part by combining BBa_K2809001, BBa_K2809002 and BBa_K2809030. This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of peroxidase 16 gene sequence, FLAG-tag for Western blot protein detection and 6xHis-tag for protein purification. It will produce the mature peroxidase 16 without Prepro-alpha-factor, and the molecular weight of enzyme should be 35.29 kilodaltons, but because of protein N-linked glycosylation, the molecular weight of enzyme will be about 37~45 kilodaltons according to the Western blot result.
Part construction in pSB1C3:
Well 1: Marker/ Well 2~4: Px16 colony1~3/ Well 6~8: Px18 colony1~3
Well 1: Marker/ Well 2~4: Lac1 colony1~3
Insert long after PCR — Px16: 1271bp/ Px18: 1289bp/ Lac1: 2036bp
Miniprep plasmid digest by EcoRI, PstI
Well 1: Marker/ Well2, 4, 6: Uncut plasmid of Px16 colony1, 2, 3/ Well3, 5, 7: Plasmid of Px16 colony1, 2, 3 after EcoRI, PstI digest/ Well8, 10, 12: Uncut plasmid of Px18 colony1, 2, 3/ Well9, 11, 13: Plasmid of Px18 colony1, 2, 3 after EcoRI, PstI digest/ Well14,16,18: Uncut plasmid of Lac1 colony1, 2, 3/ Well15,17,19: Plasmid of Lac1 colony1, 2, 3 after EcoRI, PstI digest
Vector long after digest — pSB1C3: 2052bp
Insert long after digest — Px16: 1269bp/ Px18: 1287bp/ Lac1: 2034bp
Part construction in pGAPZ A:
Well1: Marker/ Well2, 3, 4: Lac1 Colony 1, 2, 3/ Well5, 6, 7: Px16 Colony 1, 2, 3/ Well8, 9, 10: Px18 Colony 1, 2, 3/ Well11: pGAPZ A Colony
Insert long after PCR — pGAPZ A_Px16 :1377 bp/ pGAPZ A_Px18 :1359 bp/ pGAPZ A_Lac1 :2124bp
Miniprep plasmid digest by EcoRI, AgeI
Well 1: Marker/ Well2, 3, 4: Plasmid of Lac1 colony1, 2, 3 after EcoRI, AgeI digest/ Well5, 6, 7: Plasmid of Px16 colony1, 2, 3 after EcoRI, AgeI digest/ Well8, 9, 10: Plasmid of Px18 colony1, 2, 3 after EcoRI, AgeI digest
Vector long after digest — pGAPZ A: 2884bp
Insert long after digest — Px16: 1295bp/ Px18: 1277bp/ Lac1: 2042bp
Different copy number Strain select by Zeocin™ selection:
Protein expression level of each time point:
After the selection, we picked one colony from low copy number, middle copy number, and high copy number by comparing the different growth on condition of different Zeocin™ concentration plates.
Then we incubated those strains in 50ml Tube containing 5ml of YPD+100μl/ml Zeocin™ at 25℃, and set the time points every 24 hours to collect the sample after 5 times.At day 5, we centrifuged all of our samples, separated the supernatant and lysed the pellet, then detected the protein expression in cells.
Growth curve measurement:
We also calculated the growth curve by record OD595 at each time points. We incubated the different copy number strains in 50ml tube containing 5ml of YPD+100μl/ml Zeocin™ at 25℃, and measured the OD595 of sample at 12hr, 24hr, 48hr, 72hr, 96hr, and 120hr.
We supposed the growth curve was majorly controlled by the Zeocin™ resistance gene (the copy number) and influenced both of intracellular and extracellular enzymes.
In this case we supposed that the peroxidase 16 had negative influence on the growth of P.pastoris in both intracellular and extracellular. The high copy number strains grew fast in the beginning, but grew slowly as they turned into the stationary phase, thus lead to lower cell number. The low copy number strains grew slowly in the beginning, but grew slowly as they turned into the stationary phase, thus lead to higher cell number. Both copy number and enzymes influenced on the middle copy number strains, which even caused sluggish growth.
The difference between control group and pGAPZ A_Px16 strains is majorly effect by Zeocin™ resistance. Because we did not select the control, the copy number of control group is lower than pGAPZ A_Px18 strains. In other words, it caused slow growth.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 665
Illegal BamHI site found at 949
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC