Final Progress Report for COG 0229s

Alexandra Gardino

Five organisms were chosen as possible targets for protein expression for COG 0229s; Homo sapiens, Caenorhabditis elegans, Saccharomyces cerevisiae, Haemophilus influenzae, and Bacilus subtilis. The following is a protocol for making the appropriate constructs as well as the final status for each organism.

Primers were designed for each PCR product containing 3 restriction sites, 1 of which contained an initiator methionine, and 2 consecutive stop codons. Two PCR products were designed for each organism. One contains an altered C-terminus (called 1-2) and the other contains an unaltered C-terminus (called 1-3). PCR products were synthesized using the program #4 READ1. The DNA was then sent off for sequencing at the RWJMS DNA Synthesis and Sequencing Laboratory.

Accurate PCR products were then gel extracted and purified using the QIAEX II Gel Extraction protocol. The PCR products were then ligated and transformed into the holding vector pCRII-TOPO using the TOPO-TA Cloning protocol by Invitrogen. The cloning reaction was plated onto warm LB-amp plates containing X-gal for blue/white selection. The plates were grown overnight at 37 C. 3 white colonies and 1 blue colony were chosen for each PCR product to test by colony PCR to determine if there were any false positives (inaccurate white colonies) or false negatives (inaccurate blue colonies). The primers used to make the initial PCR products were used to test if the cloning reaction was a success. Positive results were grown overnight and the DNA was harvested by the QIAprep Spin Miniprep Kit protocol.

Double restiction digests were then performed to double check that the pCRII-TOPO holding vector truly contained the DNA of interest. The ATG-containing restriction enzyme (RE) and the C-terminal restriction enzyme (RE3) were chosen for the double digests. Positive results were shown for every PCR product except for B. subtilis 1-2 which was cloned into pCRII-TOPO a second time. At this time, all of the DNA was resequenced to make sure that no errors were made during the ligation and transformation process.

For those sequences that had absolutely no errors, the pCRII-TOPO vector containing the insert was transformed into XL1-BLUE (NOVA Blue) competent cells and plated onto LB plates containing ampicillin and kanamycin. For any successful transformations, overnight cultures were grown. The following day 10 l from the overnight culture was used to inoculate a 100 ml culture of LB and ampicillin and kanamycin which was then grown overnight. The DNA was harvested from this large scale prep using the QIAfilter Midi Protocol. The concentration of the midi preps were then checked on the spectrophotometer at l =260 nm.

Large scale double restriction digests were set up for preparing the expression vectors with the appropriate cuts corresponding to the restriction enzymes used for each PCR product. The DNA itself had to be cut out of the holding vector as well. Once the digest had incubated for 3 or more hours, the sample was loaded onto an agarose gel for gel extraction and purification using the QIAEX II Gel Extraction protocol. The results were then run on another gel to check the final concentration of DNA and vector.

Rapid DNA Ligation reactions were then performed to try and ligate the DNA of interest into its appropriate expression vector using a 3:1 insert to vector ratio. The hour-long ligation reaction was then transformed into XL1-Blue competent cells and plated onto LB plates containing ampicillin. In order to check if the ligation reaction was a success, either colony PCR tests or double restriction digests were performed. Expression vectors that were shown to contain the insert were then transformed into BL21-DE3 and BL21-DE3pLys competent cells and plated onto LB-amp plates to grow overnight. The vectors were then ready for expression.

Colonies were picked off of the plates to grow in overnight cultures. For any constructs that had T7 promoters (pET 14 and pET 23), 2% glucose by volume was added to the 3 ml overnight cultures. After 16 to 18 hours of growth, 200 ml of the overnight cultures were added to 4 ml of fresh LB media containing ampicillin. After 2 hours of growth at 37° C with shaking, the OD (optical density) of 1 ml of cells was checked at l =600 nm. Cells with an OD at .5 to .8 were not allowed to continue to grow. 500 l of the initial 1ml used to check the OD was spun down for a few minutes and labeled U (for uninduced sample). 1M IPTG was added to the other 3 ml of the sample to reach a final concentration of 1mM IPTG and allowed to grow for another 3 hours. 1 ml was used to check the OD of the induced samples, and 500 l was spun down and labeled I (for induced sample).

A loading dye mixture of 2xP , SDS loading dye, and BME was prepared. The cell pellets were resuspended in 100x l loading dye of their OD at 600 nm. A protein gel was poured, and a marker as well as 3 protein standards were loaded onto the gel. Both the uninduced and induced protein samples were loaded onto the gel to determine the level of expression.

Status of Project and Location of Reagents

Homo sapiens

PCR product H.sap 1-2:

Successful, ligated into pCRII-TOPO in XLI-Blue cells without any errors.

Restriction digest stocks for appropriate expression vectors:

DNA: H.sap 1-2 NcoI/HindIII

Vectors: pET23d-C

pET21d-C

pOE60-C

Successful ligation into pET23d-C and pET21d-C.

Non-successful ligation into pQE60-C

Expression of pET23d-C at 6 mg/ml

Glycerol stocks made for the completed constructs:

H.sap 1-2 pET23d-C

H.sap 1-2 pET21d-C

PCR product H.sap 1-3:

Several attempts were made to clone into pCRII-TOPO but sequencing revealed several errors in the Primer 1 (forward) region. No further experiments were conducted.

Caenorhabditis elegans

PCR product C.ele 1-2:

Successful, ligated into pCRII-TOPO in XL1-Blue cells without any errors.

Restriction digest stocks for appropriate expression vectors:

DNA: C.ele 1-2 NdeI/BamHI

Vectors: pET23c-C

PET21c-C

PCR product C.ele 1-3:

Successful, ligated into pCRII-TOPO in XLI-Blue cells without any errors.

Restriction digest stocks for appropriate expression vectors:

DNA: C.ele 1-3 NdeI/SalI

Vectors: pET14c-C

pET15c-C

pET23c-C

pET21c-C

DNA: C.ele 1-3 SacI/SalI

Vectors: pQE30

Saccharomyces cerevisiae

PCR product S.cer 1-2:

Successful, ligated into pCRII-TOPO in XL1-Blue cells without any errors.

Restriction digest stocks for the appropriate expression vectors:

DNA: S.cer 1-2 NdeI/SacI

Vectors: pET21c-A

PET23c-A

Successful ligation into pET21c-A and pET23c-A

Glycerol stocks made for the completed constructs:

S.cer 1-2 pET21c-A

S.cer 1-2 pET23c-A

PCR product S.cer 1-3:

Successful, ligated into pCRII-TOPO in XL1-Blue cells without any errors.

Restriction digest stocks for the appropriate expression vectors:

DNA: S.cer 1-3 BamHI/SalI

Vectors: pQE31

DNA: S.cer 1-3 NdeI/SalI

Vectors: pET14c-C

pET15c-C

pET23c-C

pET21c-C

Successful ligation into pET14c-C, pET15c-C, pET21c-C, and pET23c-C

Glycerol stocks made for the completed constructs:

S.cer 1-3 pET14c-C

S.cer 1-3 pET15c-C

S.cer 1-3 pET21c-C

S.cer 1-3 pET23c-C

Non-successful ligation into pQE31

Haemophilus influenzae

PCR product H.inf 1-2:

Successful, ligated into pCRII-TOPO in XL1-Blue cells without any errors.

Restriction digest stocks for the appropriate expression vectors:

DNA: H.inf 1-2 NcoI/HindIII

Vectors: pET23d-A

pET21d-A

pQE60-A

PCR product H.inf 1-3:

Successful, ligated into pCRII-TOPO in XL1-Blue cells without any errors.

Restriction digest stocks for the appropriate expression vectors:

DNA: H.inf 1-3 NcoI/SalI

Vectors: pET14d-C

PET15d-C

DNA: H.inf 1-3 BamHI/SalI

Vectors that have not been prepared:

pQE31

pET23d-C

pET21d-C

Bacilus subtilis

PCR products B.sub 1-2 and B.sub 1-3:

Several attempts were made to clone into the pCRII-TOPO holding vector but DNA sequencing revealed several errors in the Primer 1 (forward) region. New primer was ordered and sequencing errors continued. No further experiments were conducted.

 

Location of Reagents

  1. All glycerol stocks for the completed constructs are on the second shelf up from the bottom in the — 70 ° C freezer labeled Freezer 1. They are located in a white box labeled Glycerol stocks for COG 0229s in blue tape.
  2. All restriction digest stocks are in the — 20 ° C freezer labeled Freezer 3. They are located in a white box labeled COG 0229s Proteins/Vectors in red tape.
  3. All primers are located in the — 20 ° C freezer labeled Freezer 3. They are located in a white box labeled Primers for COG 0229s in pink tape.
  4. All PCR products, minipreps, and midipreps are located in the - 20° C freezer labeled Freezer 3. They are located in a white box labeled DNA for COG 0229s in green tape.

*** The cDNA for H. sapiens and C. elegans is located in this box as well!!

5.) The other genomic DNA for B. subtilis, H. influenzae, and S. cerevisiae is located

on the bottom left hand shelf of the refridgerator labeled Fridge 2.

TO WHOM EVER TAKES THIS PROJECT OVER, GOOD LUCK!!