Figures

A portion of remaining PCR product was subcloned into pTrc-His vector via TOPO-TA cloning. pTrc-His vector was transformed into E. coli, and cells were plated and incubated. Remainder of PCR product was purified via Qiagen QIAquick PCR Purification and added to a sequencing plate. Samples were sent to a core facility at Dana-Farber to be sequenced.

E. coli cells were co-transformed with pBT-p53 plasmid and a library of pTRG plasmids containing different cDNA inserts, plated onto Tet-selective medium lacking histidine, and incubated.

Induced and uninduced samples were incubated each with and without p53. Electrophoretic Mobility Shift Assay was performed on a polyacrylamide gel.

E. coli were plated on Amp-selective medium and incubated. Cells were screened for orientation of the target gene. Proper orientation was analyzed via PCR and electrophoresis of PCR product on an E-gel.

A portion of the PCR product was analyzed through electrophoresis on an Invitrogen E-gel.

Bradford assay was performed to determine protein concentration of induced and uninduced lysates.Whole cell induced and uninduced samples were obtained. Lysed induced and uninduced samples, each with SDS and without SDS, were also prepared.

STARTA colony was selected and pTRG insert (target gene) was amplified via PCR.

Sequencing results were analyzed using FinchTV and compared to human genome sequences using nucleotide BLAST.

A properly oriented TOPO-TA clone was selected, incubated and grown up. Log-phase cell cultures were either induced with IPTG or uninduced.

Induced and uninduced whole cell samples, and induced and uninduced lysate SDS samples of equal protein content were run on a polyacrylamide gel to detect induction.

END

Figure 1. Flowchart of experimental process

(A) Lane 1 contains a New England Biolabs 100 base pair ladder, Lane 7 contains the negative control (PCR reaction run with PCR reagents but no pTRG plasmid), and Lane 8 contains the sample with amplified pTRG insert(B) New England Biolabs 100 base pair ladder

A B

1500 bp

Figure 2. Gel electrophoresis of PCR-amplified pTRG insert from E. coli transformed colony

Lane 1 contains 100 base pair ladder (see Figure 2B). Lanes 7-9 contain PCR product of colonies selected from incubation of E. coli transformed with pTrc-His vectors from TOPO-TA cloning. Samples in lanes 7, 8, and 9, respectively, were amplified with pTrc-His reverse primer and pTRG forward primer.

1700 bp

Figure 3. Gel Electrophoresis of PCR-amplified pTrc-His insert for screening of proper orientation

A

B

(A) Snapshot of FinchTV chromatogram of sequenced pTRG target gene. Base pairs 100-150 are shown.(B) Snapshot of top Nucleotide BLAST results. Shown are the top alignment matches of sequenced pTRG target gene compared with all human mRNA transcripts. Base pairs 100-600 were searched in Nucleotide BLAST.

Figure 4. Chromatogram and BLAST results of PCR-amplified target gene on pTRG plasmid

0 100 200 300 400 500 600 700 800 900 10000

0.05

0.1

0.15

0.2

f(x) = 0.000204657534246575 x + 2.73972602739675E-05R² = 0.999994623771532

Absorbance vs. Protein Con-centration

Protein Concentration (µg/mL)

Ab

sorb

ance

at

59

5n

m (

AU

)

Absorbance at 595 nm of 800 g/mL and 900 g/mL protein standards incubated with Bradford reagent for 5 minutes. The line was obtained using the Best Fit Line feature in Microsoft Excel. The line’s equation was used to calculate the protein concentration of uninduced and induced cell lysates.

Figure 5. Bradford assay of absorbance vs. protein concentration

(A) Lane 6 contains a Bio-Rad Precision Plus Protein Marker. Lanes 7 and 8 contain 30 L of whole cell sample suspended in SDS dye. Transcription of the gene of interest was uninduced and induced with IPTG for lanes 7 and 8, respectively. Lanes 9 and 10 contain 41.7 g of protein from samples lysed with commercial lysis buffer and suspended in SDS dye. The gene of interest was uninduced or induced with IPTG in lanes 9 and 10, respectively.(B) Bio-Rad Precision Plus Protein Marker

Lane 1 2 3 4 5 6 7 8 9 10

B

50 kD

37 kD

Figure 6. Protein gel of uninduced and IPTG-induced E. coli samples

A

Lanes 7-9 contain 0.5 L lysate buffer, uninduced, and induced bacterial lystate, respectively, in the absence of p53. Lanes 10-12 contain 0.5 L lysate buffer, uninduced, and induced bacterial lystate, respectively, incubated with 1 L p53. All bands were detected by incubating samples with a non-radioactive, fluorophore p53 response element DNA probe.

Lane 1 2 3 4 5 6 7 8 9 10 11 12

Figure 7. Electrophoretic Mobility Shift Assay gel of target protein with p53