June - Week 3

June 19th

Experimenter : Dan Korvin, Wenchen Zhao, Zeki Ekmekci

Aim : Amplify T7 RNA polymerase gene (RNAP) and terminator gene by PCR.

Result : PCR was successful. Target bands were seen on the agarose gel.

June - Week 4

June 23th

Experimenter : Zeki Ekmekci, Wenchen Zhao, Dan Korvin

Aim : Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes by PCR. Digest terminator with EcoRI (E) and XbaI (X). Digest RNAP with E and SpeI (S).

Results : NA.

June 24th

Experimenter : Ariel Ragetli, Zeki Ekmekci

Aim : Verify DNAP, P/H, SSBP genes on agarose gel. Digest DNAP, P/H, SSBP amplicons with E and S. Ligate each digested T7 gene to digested terminator vector.

Results : DNAP, P/H, SSBP genes successfully amplified, but non-trivial amount of side products (mostly primer-dimer) appeared in DNAP and P/H lanes. Four T7 genes all digested and ligated with terminator.

June 28th

Experimenter : Wenchen Zhao

Aim : Verify the ligation results on agarose gel.

Results : Multiple bands observed (non-trivial amount of side products). Target SSBP+terminator fragment with distinctive amount observed (only one lane with decent result).

June 29th

Experimenter : Jeffrey Pea

Aim : Amplify the target gene/terminator ligated sequence using four different forward primers and same VF reversed primer. Verify the PCR products on a gel.

Results : No significant bands we were looking for observed on the gel.

June 30th

Experimenter : Tudor Lapuste

Aim : Amplify four T7 genes and terminator gene by PCR. Digest four genes using E and S.

Results : SSBP, DNAP and RNAP were amplified successfully. P/H and terminator PCR failed. PCR for P/H and terminator set up.

July - Week 1

July 2nd

Experimenter : Dan Korvin

Aim : Verify P/H + terminator PCR products on agarose gel.

Results : PCR failed for both. More PCR set up for primase/helicase.

July 3rd

Experimenter : Ariel Ragetli, Jeffrey Pea, Zeki Ekmekci

Aim : Verify PCR products for P/H on agarose gel. Amplify terminator by gradient PCR. Transform E.coli cells with terminator-containing plasmid.

Results : Terminator and P/H genes amplified.

July 4th

Experimenter : Dan Korvin

Aim : Digest terminator with E and X. Run PCR products of all four T7 genes on a gel, followed by gel extraction and purification.

Results : NA

July - Week 2

July 6th

Experimenter : Zeki Ekmekci, Wenchen Zhao, Jeffrey Pea

Aim : Double digest all four T7 genes with E and S. Run digested T7 genes and terminator on a gel, followed by gel extraction and purification. Ligate T7 genes with terminator. Transform all four constructs to E.coli

Results : NA

July - Week 3

July 10th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Verify constructs by colony PCR.

Results : Colony PCR failed. No bands were observed.

July 17th

Experimenter : Jeffrey Pea

Aim : Double digest terminator plasmid with E and X. Ligate digested T7 genes to terminator. Transformed four constructs to E.coli

Results : NA

July - Week 4

July 20th

Experimenter : Tudor Lapuste

Aim : Verify constructs by colony PCR.

Results : SSBP and RNAP constructs (gene + terminator) confirmed.

July 24th

Experimenter : Ariel Ragetli, Jeffrey Pea

Aim : Re-amplify DNAP and P/H by PCR followed by gel check. Ligate two digested amplicons to digested terminator. Transform two constructs into E.coli.

Results : DNAP and P/H were successfully amplified.

August - Week 1

August 1st

Experimenter : Wenchen Zhao, Dan Korvin, Zeki Ekmekci

Aim : Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest Promoter + RBS plasmid with E and X. Transform the ligation products to E.coli.

Results : Colony PCR failed.

August 6th

Experimenter : Wenchen Zhao, Jeffrey Pea, Zeki Ekmekci

Aim : Verify DNAP and P/H constructs (gene+terminator), SSBP and RNAP constructs (gene+terminator+RBS+promoter) using colony PCR.

Results : P/H and DNAP constructs confirmed. Bands with correct size were found on the gel after colony PCR of SSBP and RNAP constructs.

August - Week 2

August 12th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Prepare SSBP and RNAP miniprep products and send them for sequencing. Double digest P/H and DNAP constructs with E and S. Ligate digested constructs into vector digested with E and X. Transform ligation products into E.coli.

Results : NA.

August - Week 3

August 19th

Experimenter : Ariel Ragetli, Zeki Ekmekci

Aim : Verify P/H and DNAP constructs using colony PCR. Analyze sequencing results of SSBP and RNAP complete constructs.

Results : Sequencing results are negative – no promoter/RBS found in SSBP and RNAP constructs. Colony PCR result was also negative.

September - Week 1

September 4th

Experimenter : Dan Korvin, Wenchen Zhao

Aim : Double digest DNAP, RNAP and SSBP constructs with X and PstI (P). Ligate three digested genes into RBS+Promoter plasmid digested with S and P. Miniprep P/H construct.

Results : NA.

September 5th

Experimenter : Wenchen Zhao

Aim : Transform all three constructs into E.coli. Site-directed mutagenize P/H construct and transform into E.coli.

Results : NA.

September 6th

Experimenter : Anna Müller

Aim : Verify DNAP, RNAP and SSBP constructs by colony PCR.

Results : Bands with correct size observed, need to send to sequencing for further verification.

September - Week 2

September 8th

Experimenter : Anna Müller

Aim : Miniprep all cultures including SDM P/H construct with terminator and complete DNAP, RNAP and SSBP constructs

Results : NA

September 9th

Experimenter : Wenchen Zhao, Zeki Ekmekci

Aim : Send DNAP, RNAP, SSBP constructs for sequencing. Double digest P/H constructs with X and P, followed by gel check. Double digest RNAP with E and X.

Results : Gel check result was negative. No target band was observed. P/H was not mutagenized.

September 10th

Experimenter : Ariel Ragetli, Jeffrey Pea

Aim : Double digest SSBP with E and S. Double digest RNAP with E and X, followed by gel check and extraction. Ligate digested SSBP into digested RNAP vector. Transform ligation product into E.coli.

Results : NA.

September - Week 3

September 15th

Experimenter : Wenchen Zhao

Aim : Colony PCR SSBP + RNAP construct. Repeat SDM of P/H construct.

Results : Colony PCR failed.

September 16th

Experimenter : Wenchen Zhao

Aim : Re-run colony PCR of SSBP and RNAP construct.

Results : Colony PCR result was positive.

September 17th

Experimenter : Wenchen Zhao

Aim : Miniprep SSBP + RNAP construct, double digest with E and S. Double digest DNAP with E and X, followed by gel verification and extraction. Ligate digested DNAP with digested SSBP + RNAP construct. Transform SDM P/H plasmid.

Results : NA.

September 18th

Experimenter : Wenchen Zhao

Aim : Colony PCR SDM P/H. Double digest P/H with X and P.

Results : Colony PCR failed.

September 19th

Experimenter : Wenchen Zhao

Aim : Repeat colony PCR of SDM P/H. Miniprep DNAP, RNAP for sequencing.

Results : Colony PCR results were positive.

September - Week 4

September 21st

Experimenter : Zeki Ekmekci

Aim : Double digest 3-gene assembly with E and P. Double digest SDM P/H with X and P. Ligate digested P/H to digested RBS plasmid. Transform ligation product to E.coli.

Results : NA.

September 23rd

Experimenter : Wenchen Zhao

Aim : Colony PCR P/H construct followed by gel check.

Results : Colony PCR result was positive.

September 24th

Experimenter : Wenchen Zhao

Aim : Analyze sequencing results for DNAP, SSBP and RNAP constructs. Miniprep SDM P/H and send it for sequencing.

Results : Sequencing results were positive. DNAP, SSBP and RNAP constructs were successfully assembled.

October - Week 1

October 1st

Experimenter : Wenchen Zhao

Aim : Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.

Results : Sequencing result was negative. Promoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.

October 2nd

Experimenter : Wenchen Zhao

Aim : Double digest SDM P/H with X and P. Double digest promoter/RBS plasmid with S and P. Ligate digested P/H into digested promoter/RBS plasmid. Transform the ligation product into E.coli.

Results : NA

October 3rd

Experimenter : Wenchen Zhao

Aim : Verify P/H constructs by colony PCR.

Results : Colony PCR result was positive. Target band was observed on the gel.

October - Week 2

October 4th

Experimenter : Ariel Ragetli

Aim : Verify P/H constructs by colony PCR.

Results : Colony PCR result was positive. Target band was observed on the gel.

October 7th

Experimenter : Jeffrey Pea

Aim : Miniprep P/H plasmid and send it for sequencing.

Results : NA

October 8th

Experimenter : Jeffrey Pea

Aim : Double digest P/H with E and X. Double digest 3-gene assembly with E and S. Ligate digested P/H into digested 3-gene assembly. Transform the ligation product to E.coli.

Results : NA

October 9th

Experimenter : Jeffrey Pea

Aim : Verify 4-gene assembly by colony PCR followed by PCR.

Results : Colony PCR result was positive. 4-gene construct was assembled.

October 10th

Experimenter : Wenchen Zhao

Aim : Analyze sequencing result for P/H plasmid.

Results : Sequencing result was negative. There was a ~200bp deletion in the middle of P/H gene. Also there was a 2bp deletion at the beginning of the gene, causing a frameshift mutation

October - Week 3

October 14th

Experimenter : Wenchen Zhao

Aim : Verify protein expression of 4-gene assembly on SDS-PAGE.

Results : DNAP and SSBP protein was successfully expressed. P/H expression was not detected likely due to the mutation. RNAP expression was also not detected.