Part:BBa_K2809021

peroxidase 18

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_K2809020. 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 18 gene sequence, V5-tag for Western Blot protein detection and 6xHis-tag for protein purification. It will produce the mature peroxidase 18 without Prepro-alpha-factor, and the molecular weight of enzyme should be 33.64 kilodaltons. However, because of protein N-linked glycosylation, the molecular weight of enzyme will be about 38~40 kilodaltons according to the Western blot result.

Part construction in pSB1C3:

PCR result

Yeastern Biotech 1Kb YEA Ladder DNA Marker III

First gel:

Well 1: Marker/ Well 2~4: Px16 colony1~3/ Well 6~8: Px18 colony1~3

Second gel:

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:

PCR result

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:

After transform the vector into Pichia pastoris SMD1168, we selected the strains by growth the single colony on different Zeocin™ concentration plates.

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 centrifuges all of our samples, separated the supernatant and lysed the pellet, then detected the protein expression in cells and soup by western blot.

western blot (soup) of high copy strain

western blot (protein extraction from pellet) of high copy strain

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 OD ₅₉₅ samples 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 on both of intracellular and extracellular enzymes.

In this case we supposed that the peroxidase 18 had negative influence on growth of P. pastoris in high concentration, which caused a slow phase of growth at 72hr to 96hr. After the enzymes degraded intracellular, the growth curve would increase again.

The difference between control group and pGAPZ A_Px18 strains was majorly effected by Zeocin™ resistance. Because we did not select the control group, the copy number of control group is relatively lower than pGAPZ A_Px18 strains. In other words, it caused slow growth.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]