Team:HKUST/Parts

• Project
  • Description
  • Design
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• Dry Lab
  • Protein Structure Pretiction
  • Molecular Dynamics Simulation
  • Color Mixing
• Wet Lab
  • Description
  • Cloning
  • Expression & Purification
  • Thread Formation
  • Color Mixing
  • Draw-Processing
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Parts

• Basic Parts for IF Production

• Promoter Comparisons

• Expression Methods

• Linking Chromoprotein at Different Positions

• Color of Chromoproteins

Overview

Our project revolves around synthesizing a type of intermediate filament proteins which can be used for weaving fabrics, so our biological circuits are designed to facilitate strong expression and secretion of the target protein subunits in order to increase product yield. We hope our circuit can be applied to the manufacturing process of our protein production.

The two (2) protein subunits (α Keratin: BBa_K3581000 and γ Keratin: BBa_K3581001) are enclosed in two plasmids and then expressed separately using E. coli (BL21) as a chassis. The genes of interest are geared with a strong-promoter-strong-RBS composite part (BBa_K880005) and winded up with a commonly used terminator (BBa_B0015). Alternative promoter (T7 promoter: BBa_I712074), RBS (BBa_B0030), and terminator (T7 terminator: BBa_B0016) are used to replicate previous experimental data in order to evaluate and compare protein expression rates.

Chromoproteins of three different colors (Blue: BBa_K3581004, Red: BBa_K3581005 and Yellow: BBa_K3581013) are linked to the N or C-terminus of the subunits for pre-dyeing purposes.

An affinity tag (6xHis-tag: BBa_K3581002) is added after the chromoproteins to facilitate purification and characterization. A TEV protease cleavage site (amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)) is placed upstream of the 6xHis-tag to facilitate tag removal.

A short signal peptide (HlyA Signal Peptide: BBa_K3581003) is incorporated at the C-terminus of both circuits to induce extracellular secretion of expressed proteins.

A high copy plasmid will be used to obtain better yield of protein subunits (pSB1C3: high copy number plasmid carrying chloramphenicol resistance).

Since we will be switching different parts of the circuit to suit our experimental purposes such as changing chromoproteins for colour or promoters for product yield, we will be using restriction enzymes Acc65I, AscI and HindIII to do so.

Acc65I recognition sites will be added in between the RBS and protein subunit, a separate chromoprotein part will be ordered and it is flanked by two Acc65I recognition sites in order for the protein to be digested and ligated into the desired sequence and linked to the N terminus.

AscI recognition sites will be added in between the affinity tag and protein subunit, a separate chromoprotein part will be ordered and it is flanked by two AscI recognition sites in order for the protein to be digested and ligated into the desired sequence and linked to the C terminus.

HindIII will be flanking the signal peptide part. As our experiment will involve secreting the protein as an inclusion body or extracellular secretion, so we included HindIII cut site to digest the signal peptide part when our experiment requires us to perform extracellular secretion.

Parts name/included parts name/parts no.	Constructs descriptions & Applications in our project (Link to page)	Diagram of constructs (& Links)
Basic IF Production
T7 IF Alpha & Gamma without signal peptide	This construct is designed for expressing and producing our alpha and gamma subunits using the T7 promoter and terminator set. T7 is used because we want to try and replicate previous experiment results.
K88 IF Alpha & Gamma without signal peptide	This construct is designed for expressing and producing our alpha and gamma subunits using the BBa_K880005 composite part. BBa_K880005 is used because we want to try and maximize protein secretion.
Promoter Comparisons
K88 & T7 without signal peptide	This construct is designed to compare the strengths and differences between the two promoter sets we have used for IF production.
Expression Methods Comparisons
K88 & T7 without signal peptide	This construct is designed to compare different expression methods of the proteins. Without a signal peptide means the protein will be secreted as inclusion bodies and we want to determine the yield difference.
K88 & T7 with signal peptide	This construct is designed to compare different expression methods of the proteins. With a signal peptide means the protein will be secreted extracellularly and we want to determine the yield difference.
CP linking in different terminal Comparisons
T7 IF Alpha & Gamma ,CP in N-terminus	This construct is designed to insert chromoproteins at the N terminus of the IF, in front of the protein subunit, using T7 sets. Comparing the insertion in N or C terminus will help us determine which has the better dyeing effect.
T7 IF Alpha & Gamma ,CP in C-terminus	This construct is designed to insert chromoproteins at the C terminus of the IF, in front of the protein subunit, using the T7 sets. Comparing the insertion in N or C terminus will help us determine which has the better dyeing effect.
K88 IF Alpha & Gamma ,CP in N-terminus	This construct is designed to insert chromoproteins at the N terminus of the IF, in front of the protein subunit, using BBa_K880005. Comparing the insertion in N or C terminus will help us determine which has the better dyeing effect.
K88 IF Alpha & Gamma ,CP in C-terminus	This construct is designed to insert chromoproteins at the C terminus of the IF, in front of the protein subunit, using BBa_K880005. Comparing the insertion in N or C terminus will help us determine which has the better dyeing effect.
Different CP Colours Collections
Primary Colour
Red, Yellow, Blue , each colour has 2 Chromoproteins	This construct consists of the primary colours of chromoproteins, red, yellow and blue. Each colour comes with 2 chromoproteins.
Secondary Colour
Orange, Green , Purple, each colour has 1 CP	This construct consists of the secondary colours of chromoproteins, orange, green and purple. Each colour comes with 1 chromoprotein.