Difference between revisions of "Part:BBa K1955008"

J04500 is a LacI inducible promoter, so it can be induced by IPTG to activate the promoter for transcription. We picked two colonies of pSB1C3-J04500-HA and pSB1A2-J04500-GFP and incubated in 4ml LB broth containing antibiotics at 37℃, 250rpm for overnight. Inoculate 4ml fresh LB supplemented with antibiotics with 20μl of overnight cultures. Shake cultures at 37℃, 250rpm for 3hr. Split cultures into two (2ml each) and add 2μl 1M IPTG to one of the tube to reach 1mM IPTG for induction. The other tube will be the uninduced control. Shake both tubes for another 3hrs at 37℃, 250rpm.

For the pSB1C3-J04500-HA, their induction can be checked by Coomassie blue or Western blot analysis. Transfer cultures from both tubes to two Eppendorf tubes and centrifuge bacteria at max speed for 3min. Discard the supernatant, and resuspend each pellet in 100μl 1x SDS sample buffer by pipetting. Fit cap-guards onto Eppendorf tubes and boil the samples at 100℃ for 10min. Run SDS-PAGE to check the expression of desired protein.

Conduct the Western blot analysis, we successfully recognize the HA protein (approximately 62.3kd) in our two colonies (JH1 and JH2) when induced by IPTG (Fig. 1A).

To check the feasibility of J04500, we also built up a construct containing J04500 and GFP. After induction of 3hrs, the induced bacteria turn fluorescent green. In contrast, the uninduced bacteria did not appear to have any color change. (Fig. 1B)



Fig. 1. Protein expression of pSB1C3-HA and pSB1A2-GFP.

The day before induction, two different colonies were picked up from each construct into 4ml LB broth at 37℃, 250rpm for overnight. Inoculated 4ml fresh LB broth with 20μl of overnight cultured at 37℃, 250rpm for 3hrs. Splited into two tubes(each 2ml), and added 2μl 1M IPTG to one tube as the induced group, the other tube without IPTG was the uninduced group. IPTG induction for 3~4hrs.

(A) After IPTG induction, centrifuged bacteria at max speed. Discarded the supernatant, lysed the cell with 100μl 1x SDS sample buffer. Western blotting to check the desired gene expression. In fig. 5A, His-tag HA serves as positive control to validate that the antibody can work normally. We also transform pUC-19 into BL21 and follow the induction protocol as the negative control.

(B) IPTG induction of pSB1A2-J04500-GFP to verify the LacI inducible promoter. The GFP fluorescence can be observed easily by bare eye.

Used EcoRIHF and XbaI to cut pSB1C3-HA and pSB1A2-GFP. Due to the short gap between EX restriction cutting site, it is hard to observe double band in the double enzyme digestion.

The whole length of pSB1C3-HA is approximately 3800bp, and pSB1A2-GFP is 3000bp. By double enzyme digestion, all plasmids can be digested into one band (linear form) on the DNA gel.



Fig. 2.　Restriction enzyme cutting of pSB1C3-HA and pSB1A2-GFP.

Using EcoRIHF and XbaI with cutsmart enzyme buffer reaction for 3hr. Run a TAE gel for 45 min.

Utilized EcoRIHF and SpeI to digest pSB1C3-J04500, and served as the insert of ligation. J04500 is a 220bp DNA sequence, therefore a 2100bp (pSB1C3) and 220bp band(J04500) can be observed.



Fig. 3. Restriction enzyme cutting of pSB1C3-J04500. Using EcoRIHF and SpeI with cutsmart enzyme buffer reaction for 3hr. Run a TAE gel for 45 min.

Using EcoRIHF and XbaI with cutsmart enzyme buffer reaction for 3hr. Run a TAE gel for 45 min.

According to the calculation of the ligation protocol, we can ligate pSB1C3-HA and pSB1A2-GFP with J04500. After ligation, transform 5μl sample into DH5α competent cell.



Fig. 4. Transformation of the ligation samples. After ligation overnight, transform 5 μl samples into DH5α competent cell following the transformation protocol. Pictures show the colonies of pSB1C3-J04500-HA and pSB1A2-J04500-GFP onto LB plate.

With the primer for prefix and suffix, we easily picked up colonies and conducted colony PCR to check the transformation result. The colonies that had successfully insert J04500 into pSB1C3-HA, showed the band size of 220+1700bp (Fig. 4A). As for the negative control, we used pSB1C3-HA(1700 bp). The successful transformed pSB1A2-GFP inserted with J04500 showed the band size of 220+720 bp(Fig. 4B.) and used pSB1A2-GFP as our negative control.



Fig. 5. Colony PCR check of the insertion of J04500 in pSB1C3-HA and pSB1A2-GFP with Taq DNA polymerase and primer for prefix and suffix. (A) Check the insertion of J04500 into pSB1C3- HA, pSB1C3- HA were as the negative control. (B) Check the insertion of J04500 into pSB1A2-GFP, pSB1A2-GFP was as the negative control.