IGEM:Hong Kong HKUST/Investigations/ Plasmid Cloning of GFP Generator Using Standard Assembly
Plasmid Cloning of GFP Generator Using Standard Assembly
Soo Kyung SHIN
Leona Long Ching LAI
Cynthia Chui Shan KWOK
Maksud, Md. MAHIAN
Janice Man Jing WONG
Gary Ka Wing YUEN
Joyce On Ki WONG
Karen Kar On CHENG
Kaitlin Hao Yi CHAN
Danson Shek Chun LOI
The aim of this practical was to construct a recombinant plasmid - pSB1A2 containing constitutive promoter (BBa_J23110) and GFP generator (BBa_E5501), using Standard Assembly. Restriction enzyme digestion, gel extraction, and ligation were used to make the recombinant plasmid. The recombinant plasmid was transfected into competent E.coli cells which were then plated onto agar plates containing ampicillin. Colony PCR was used to check the plasmids retrieved from the transformed cells. The results showed that desired ligation product pSB1A2-BBa_E5501-BBa_J23110 was successfully transformed into 2 out of 12 of the selected colonies.
The aim of this practical was to create a recombinant plasmid containing GFP generator. The Biobricks used in this practical was BBa_E5501 and BBa_J23110. BBa_E5501 contains a green fluorescent gene BBa_E0040 and a RBS BBa_B0032, while BBa_J23110 is a constitutive promotor. By Biobricks Standard Assembly, the two parts were combined and transformed into E.coli and resulted in cells that produce GFP.
Methods and Materials
Table 1. BioBricks used in this investigation.
|BBa_J23110||35||pSB1A2||RFP Producing Constitutive Promoter|
|BBa_E5501||771||pSB1C3||RBS (BBa_B0032) and GFP (BBa_E0040)|
Figure 1. Standard assembly of BBa_E5501 and BBa_J23110. As illustrated in Figure 1, pSB1C3-BBa_E5501 was cut by Xbal and Pstl-HF and isolated as an insert, while pSB1A2-BBa_J23110 was cut by the restriction enzyme Spel-HF and Pstl-HF and serve as a vector in the ligation. After the restriction digestion, the interested fragments were firstly purified through gel purification, and then mixed and ligated using T4 ligase. The end product of the ligation was transformed into the competent cell.
1. Restriction enzyme digestion pSB1C3_BBa_E5501 was digested with XbaI and PstI-HF and pSB1A2_BBa_J23110 was digested with SpeI-HF and PstI-HF. 1000ng of each plasmid was used for digestion. 0.2μl of each enzyme was used with 1.8μl of Cutsmart buffer. The mixture was topped up to 18μl and incubated for 1 hour at 37°C.
Table 2. Restriction Enzyme Digestion Recipe
|DNA Concentration (ng)||1000||1000|
|Sample DNA Concentration (ng/μL)||101.1||141.2|
|DNA Amount (μL)||7.08||9.89|
|CutSmart Buffer (μL)||1.8||1.8|
2. Gel electrophoresis, Extraction and Purification, and Nanodrop test Gel electrophoresis was performed using 1% agarose gel at 130V for 28 minutes. Then a gel photo was obtained. Based on the result of gel electrophoresis, the required bands were extracted and purified according to the protocol. Nanodrop machine was used to check the purity of the DNA by calculating the DNA concentration, protein contamination and salt contamination.
Table 3. Nanodrop result
3. Ligation of the digested BBa_E5501 and BBa_J23110 4.17μl of digested BBa_J23110 and 9.41μl of digested BBa_E5501 were ligated with 1μl T4 ligase in 1μl 10x DNA ligase buffer. The reaction mixture was topped up to 10μl with ddH2O. A set of negative control was also conducted, with T4 ligase left out in the reaction mixture.
The ligated products and competent cells were mixed and put on ice for 20 minutes. Then, heat shock (42°C, 90 seconds) and cold shock (on ice, 2 minutes) were performed. 1000μL LB broth was added to the tubes and incubated at 37°C for 1 hour. After that, the content of each tubes were spread on agar plate with ampicillin and incubated overnight.
5. Colony PCR
2μL of template DNA from the colonies were added in to mastermix containing 15-fold of 2μL 10x Taq reaction buffer, 5μL 1mM dNTPs, 0.5μL 10μM forward primer VF2 and backward primer VR, 0.125μL Taq DNA polymerase and topped up to 20μL with ddH2O. The mixture was first denatured at 95°C for 3 minutes, then 24 cycles of denaturation (95°C, 30s), annealing (55°C, 60s) and extention (68°C, 1.5min). The final extention was carried out at 69°C for 4.5 minutes and held at 4°C. Gel electrophoresis was done with the samples.
Inoculation was done to the samples showing the desired bands from the result of gel electrophoresis. 5mL of LB containing ampicillin was added to the saved samples and incubated overnight.
Miniprep was carried out according to the MiniPlus DNA Extraction Kit protocol. Then the DNA purity was tested by Nanodrop.
8. Restriction check
Restriction check is not carried out in this practical since the the time was not enough.
Figure 2. pSB1A2-BBa_J23110-BBa_E5501 candidates after Colony PCR. The result was analyzed on a 1% agarose gel pre-stained with 1x Midori Green. 6 μl of 1Kb Plus DNA Ladder was used as size marker. Arrows indicate the correct band size of 1018bp.
As shown in figure 2, only 2 samples (sample 3 of set 1 and sample 1 of set 2) show desired bands. This shows that the insert in set 1 sample 3 and set 2 sample 1 should be the correct insert.
Only 2 of the 12 samples show desired bands in the gel electrophoresis for colony PCR. Some of the reason for other samples not showing any bands might be that sample diffuses out of the well, which causes dilution of samples so the concentration was not enough for bands to be shown. Another reason would be that the inserts in the samples are not correct.
The sample should be loaded as fast as possible to prevent diffusion of samples out of the well.
With reference to the result of colony PCR, the ligation product of BBa_E5501 and BBa_J23110 in the plasmid backbone pSB1A2 was successfully obtained. For further verification, restriction check should as well be performed from which a consistent result is expected to be obtained.