Team:NUS Singapore-Sci/GLB Logbook
Reporter System
GLB1
1. Cloning of the GLB1 from Genescript Plasmid
GLB1 is amplified from the template (Genscript plasmid, pCDNA3.1+C(-K)-DYK) using Q5 DNA polymerase (NEB) via PCR (Figure.1). The PCR protocol used is shown below, the primers used are GLB-HindIII-F and GLB-KpnI-R with annealing temperatures of 56℃, 57℃ and 58℃, and extension time of 1 min. The two primers used during PCR amplification also introduced additional HindIII and KpnI restriction sites at the front and back of GLB1 gene for downstream cloning. This GLB1 construct is addressed as GLB1 WT in subsequent experiments and this construct will be cloned into pEGFP-C1 for mammalian expression and pSB1C3 for parts submission.
After PCR amplification, GLB1 WT is digested with restriction enzymes HindIII and KpnI, followed by ligation into pEGFP-C1 plasmid for mammalian expression and transformation into DH5α. Twelve colonies were picked for colony PCR using Taq polymerase to ascertain the success of bacterial transformation (Figure. 2). GLB-HindIII-F and GLB-KpnI-R are used as primers for colony PCR, with an annealing temperature of 57℃ and extension time of 2 min. Figure 2 shows that colonies 1, 8 and 10 have the right insert size (approximately 2218 bp). Colonies 1, 8 and 10 were then inoculated in overnight LB cultures to isolate the plasmid and the plasmids were verified via restriction digest.
2. Production of GLB1 mutant plasmid
Two sets of point mutations were introduced into GLB1 WT for the construction of the pEGFP-GLB1 reporter system. The first point mutant was to change the amino acid (I389->P389). This (I-> P) mutation results in a 99.5% reduction in GLB gene expression. The second point mutant was created by substituting the amino acid at position 107 from phenylalanine to leucine (F107->L107), leading to 1.7% of WT ꞵ-galactosidase expression.
Mutants were created by introducing mutations into GLB1 WT using PCR based mutagenesis, with CCGF1, CCGR1, TCTF, TCTR, GLB-HindIII-F and GLB-KpnI-R primers (Figure. 3). Four different annealing temperatures: 69℃, 70℃, 71℃ and 72℃ were used. An extension time of 1 min was used for all four constructs. All bands were extracted to create their respective mutants. All reactions, except lane 12, produced the desired DNA bands. Lanes 1-4 was amplified by CCGF1 and GLB-KpnI-R to produce a band size of ~1034 bp.
Mutants were created by introducing mutations into GLB1 WT using PCR based mutagenesis, with CCGF1, CCGR1, TCTF, TCTR, GLB-HindIII-F and GLB-KpnI-R primers (Figure. 3). Four different annealing temperatures: 69℃, 70℃, 71℃ and 72℃ were used. An extension time of 1 min was used for all four constructs. All bands were extracted to create their respective mutants. All reactions, except lane 12, produced the desired DNA bands. Lanes 1-4 was amplified by CCGF1 and GLB-KpnI-R to produce a band size of ~1034 bp.
Subsequently, gel bands extracted from lanes 1-8 were put together to obtain GLB1 CCG-mut (Figure. 4). DNA extracted from lanes 9-16 were put together to obtain GLB1 TCT-mut (Figure. 5). Four annealing temperatures: 69℃, 70℃, 71℃ and 72℃ were used to create both mutants during PCR. Both mutanted genes were cloned into pEGFP-C1 plasmid.
GLB1 mutants were then transformed into DH5α and isolated for DNA sequencing. Sequencing results showed that both GLB1 mutants have the correct sites mutated. Subsequently, GLB1 mutants were transfected into HEK293T cells for characterization. Concurrently, illegal sites which included 3 PstI sites and an EcoRI site in the GLB1 mutants were also removed by PCR using the respective primers (the list of primers is available here).
3. Cloning of GLB1 and mutants into pSB1C3
Both the Biobrick prefix and suffix sites were added into EGFP-GLB1 insert by PCR and then cloned into pSB1C3 for parts submission. The prefix and suffix sites were introduced into all EGFP-GLB1 variants using PCR based mutagenesis with EcoRI Prefix F and SpeI Suffix R primers (Figure. 6). An annealing temperature of 69℃ and an extension time of 1 min were used for all three variants. All bands of size 2935 bp were excised and purified for cloning into pSB1C3 vector subsequently.
4. Checking expression of Wildtype and mutant GLB1 in HEK293T
Cultured HEK293T mammalian cells were transfected with 2 ug of GLB1 construct, GLB1 with TCT and CCG mutant plasmids in a 6 well plate, and expressed for 24hrs. Cells were harvested in cold 1X PBS using cell lifter, centrifuged and 1) lysed in 1% triton in PBS or Promega passive lysis buffer, and incubated on ice for 20 minutes, or 2) subjected to 3 freeze-thaw cycle using dry ice. The cell lysate was centrifuged at 14000 rcf for 10 minutes at 4oC to remove cell debris. Equal amount of cell lysates were loaded onto an SDS-PAGE gel, transferred to a nitrocellulose membrane, and blotted for FLAG-tag, EGFP and beta-actin (detailed protocol can be found here). The results of the western blot is shown in Figure 8. Since Promega passive lysis buffer gave the least number of unspecific bands, it was used as the lysis buffer of choice for the subsequent experiments.
5.β-Galactosidase Assay in HEK293T cell lysates
HEK293T were cultured in 100mm dishes and transfected with plasmid expressing the WT-GLB1 and the TCT and CCG mutant plasmids. Cells were harvested 24hr post transfection and lysed in 100ul Promega Passive Lysis buffer. 25ul of lysate was used in ONPG beta galactosidase assay while 10ul of lysate was used in dot blot activity assay using X-gal as substrate. The detailed protocol can be found here. For ONPG assay, enzymatic activity was quantified using absorbance at 420 nm (Table 1, Figure 9). For X-gal assay, a picture was taken after 24hr of incubation (Figure 10). In both assays, only the positive control (E. coli cells expressing pGEMT plasmid incubated with IPTG) show beta galactosidase activity.
| Sample Name | Abs1 | Abs2 |
|---|---|---|
| Non-transfected | 0.291 | 0.202 |
| EGFP-GLB1 WT | 0.200 | 0.202 |
| EGFP-GLB1 CCG | 0.130 | 0.128 |
| EGFP-GLB1-TCT | 0.135 | 0.132 |
| 6hr IPTG induction | 1.307 | 1.304 |
| 24hr IPTG induction | 1.259 | 1.252 |