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Team:Hong Kong HKUST/notebook/mod4
From 2013.igem.org
Glyoxylate Shunt Module's Notebook
June 2013
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
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
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
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 Pef-1alpha-pSB1C3
Extraction of Pef-1alpha for Biobrick
Extract out more pSB1C3 from BBa_J04450
Digestion of Pef-1alpha with EcoRI and PstI
Ligation of Pef-1alpha with pSB1c3
Transformed Pef-1alpha-pSB1C3 construct
Screen colonies and do digestion check
Weird bands show up, do colony PCR to confirm whether Pef-1alpha is ligated
Colony PCR showed that Pef-1alpha is there
Colony PCR with VF2 and VR primers didn’t give any result
August 2013
Practice Gibson Assembly using known DNA and Gibson assembly mix
Extract MLS, Pef-1alpha, aceB and polyadenylation sequence using PCR
Help Chloe do digestion check of aceA-pCMV
aceA-pCMV construct confirmed
Redo making Pef-1alpha-pSB1C3 construct
Create PMT construct for western blotting training
PMT construct made proceed with western blotting:
Build construct for Pef-1alpha characterization
Pcmv – GFP, Pef-1alpha – GFP construct, Pcmv – mCherry, Pef-1alpha – mCherry constructs
Individual DNA was extracted using PCR
Assembled using Gibson Assembly
Troubleshooting for Pef-1alpha characterization constructs
Use Gibson Assembly to make Pef-1alpha – pSB1C3 for submission
September 2013
Give Pcmv-aceA to group 1 to transfect
Pef-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
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 Pef-1alpha characterization: Pef-1alpha – GFP – polyadenylation sequence – pSB1C3
Improvement of existing Biobrick: J176171 primer designs to extract GFP with engineered complementary sequence of J176171
Extract GFP in RFC10 and RFC25 format
Heat sensitivity test of J176171 at temperatures range from 40 to 50 degrees Celsius for Gibson Assembly
