Part:BBa_K2809011

laccase 1

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_K2809010. This part contain a Kozak sequence for initiating and enhancing translation, Prepro-alpha-factor as a signal peptide for secretion, codon-optimized version of laccase gene sequence, HA tag for Western Blot protein detection and 6xHis-tag for protein purification. It will produce the mature laccase 1 without Prepro-alpha-factor, and the molecular weight of enzyme will be 63.65 kilodaltons.

Part construction in pSB1C3:

PCR result

Yeastern Biotech 1Kb YEA Ladder DNA Marker III

Frist 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 250ml flask containing 50ml of YPD at 30℃, collected the sample at day 4, and centrifuged our samples of each copy number, separated the supernatant and the pellet, then detected the protein expression in soup by western blot.

western blot (protein extraction from pellet) of high copy strain, middle copy strain, high copy strain at 96hr

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 on both of intracellular and extracellular enzymes.

In this case we supposed that the laccase 1 had not influence on P. pastoris, and the difference between control group and pGAPZ A_Lac1 strains was majorly effected by Zeocin™ resistance.

Because we did not select the control, the copy number of control is relatively lower than pGAPZ A_Lac1 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]