Team:Hong Kong HKUST/notebook/mod4

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  3. Module 3







Glyoxylate Shunt Module's Notebook

June 2013

Week 4


Design primers for aceA, aceB, MLS and EF1a extraction
Mini prepped pCMV/myc/mito
Extracted BW25113 genome
PCR out aceA and aceB for Biobrick
PCR out MLS for fuse with aceA and aceB
PCR out aceA and aceB to fuse with MLS


July 2013

Week 1

 

Transformed iDUET101
Mini prepped transformant and do digestion check
Make glycerol stock out of iDUET101
Extract out Psb1C3 from BBa_J04450 using gel extraction with XbaI and PstI
Extract E. coli BW25113 genome

 

Week 2

 

Ligation of aceA and aceB with pSB1c3
Transformed aceA-pSB1C3 and aceB-pSB1C3
Screen out colonies for aceA-pSB1C3 and aceB-pSB1C3 and do digestion check
aceB-pSB1C3 confirmed and proceed with sequencing
aceB-pSB1C3 partial sequence confirmed

 

Week 3

 

Extracted out more Psb1c3 from BBa_J04450
PCR out aceA with standard prefix and suffix
Digestion of aceA with XbaI and PstI
Ligate with pSB1C3
Screen colonies and do digestion check
aceA-pSB1C3 confirmed and proceed with sequencing
aceA-pSB1C3 partial sequence confirmed
Help Chloe do digestion check for EF1a-pSB1C3

 

Week 4

 

Extraction of EF1a for Biobrick
Extract out more pSB1C3 from BBa_J04450
Digestion of EF1a with EcoRI and PstI
Ligation of EF1a with pSB1c3
Transformed EF1a-pSB1C3 construct
Screen colonies and do digestion check
Weird bands show up, do colony PCR to confirm whether EF1a is ligated
Colony PCR showed that EF1a is there
Colony PCR with VF2 and VR primers didn’t give any result

 

August 2013

Week 1

 

Practice Gibson Assembly using known DNA and Gibson assembly mix
Extract MLS, EF-1alpha, aceB and polyadenylation sequence using PCR

 

Week 2

 

Help Chloe do digestion check of aceA-pCMV
aceA-pCMV construct confirmed
Redo making EF1a-pSB1C3 construct
Create PMT construct for western blotting training
PMT construct made proceed with western blotting:

 

Week 3

 

Build construct for EF-1alpha characterization
CMV – GFP, EF-1alpha – GFP construct, CMV – mCherry, EF-1alpha – mCherry constructs
Individual DNA was extracted using PCR
Assembled using Gibson Assembly

 

Week 4

 

Troubleshooting for EF-1alpha characterization constructs
Use Gibson Assembly to make EF-1alpha – pSB1C3 for submission

 

September 2013

Week 1

 

Give aceA-pCMV to group 1 to transfect
EF-1alpha – pSB1C3 constructed
Digestion checks and sequenced
Improvement of existing Biobrick: J176171 primer designs for J176171 to engineer with standard prefix in RFC10 and RFC25 formats
Found that J176171 are heat sensitive and degrades after denaturation

 

Week 2

 

Receive transfected  HEK293 cell from Group 1
Do western blotting with mouse anti c-myc  antibody as primary antibody and anti-mouse HRP as secondary antibody
Western blotting showed that aceA is expressed in HEK293 cell in contrast to the negative control
Build construct for EF-1alpha characterization: EF-1alpha – GFP – polyadenylation sequence – pSB1C3
Improvement of existing Biobrick: J176171 primer designs to extract GFP with engineered complementary sequence of J176171

 

Week 3

 

Extract GFP in RFC10 and RFC25 format
Heat sensitivity test of J176171 at temperatures range from 40 to 50 degrees Celsius for Gibson Assembly

 

Week 4
Content 1
Week 5
Content 1