Part:BBa_K2298000
Human basic fibroblast growth factor (bFGF)
Biology and Usage
Fibroblast growth factors(FGFs), originally known for their capability of promoting fibroblast proliferation, are a family of growth factors that has shown great potentials on tissue repair. Human FGFs consist of 22 members. FGFs exert their roles by binding to the transmembrane tyrosine kinase receptors, FGF receptors(FGFRs), therefore triggering downstream signaling cascades including RAS/MAP Kinase pathway, PI3 Kinase/AKT pathway and PLC-gamma pathway. The signaling then stimulates a wide range of cellular responses, such as cell proliferation, cell migration, cell differentiation and angiogenesis. (see review【1】)
Due to the ability to induce fibroblast proliferation and angiogenesis, FGFs have been studied extensively in terms of tissue repair in diverse kinds of tissue, with basic fibroblast growth factor(bFGF) being the most used FGF in wound healing(see review【1】). bFGF is thought to be an initiator of the wound healing process, reaching its highest concentration soon after injury and then declining to serum level【2】. bFGF may also possess the anti-scarring effect during wound healing【3】, which makes it an potential candidate for scar-free healing applications.
Design Considerations
The nucleotide sequence of bFGF mRNA was retrieved from NCBI nucleotide database(NCBI Reference Sequence: NM_002006.4:465-932), and synthesized by IGE Biotechnology LTD. Biobrick prefix and suffix was added by PCR using the following primers,
bFGF prefix: 5’ CGGAATTCGCGGCCGCTTCTAGACCATGGCAGCCGGGA 3’
bFGF suffix: 5’ AACTGCAGCGGCCGCTACTAGTAGATCCCGTTGCAACCGC 3’
and ligated onto the pSB1C3 plasmid backbone obtained from digestion of interlab test device 1(Part:BBa_J364000). The ligation was verified by PCR using VF2 and VR as primers, and was further comfirmed by Sanger sequencing by IGE Biotechnology LTD using VF2 as the forward primer.
Figure 4: Sequencing result of pSB1C3-bFGF, using VF2 as primer
Note that this part comes in the absence of the stop codon at the end of the sequence, hence this part should be cloned into vectors with pre-existing stop codon, fused with other protein with stop codon, or added a stop codon using PCR prior to use.
Although this sequence start with ACC instead of a canonical start codon ATG, the starting A can still form a complete XbaI site with BioBrick prefix for coding sequence.
2-step PCR
When it comes to constructing BioBricks, it is essential to add both BioBrick prefix and suffix However, both prefix and suffix is 22bp in length with high GC content, resulting in primers over 40bp in length, accompanied by extremely high Tm value. PCR reactions with such primers are likely to yield no intended products. Last year, the team SYSU-CHINA proposed to use shorter primers with only XbaI and SpeI restriction sites to solve this problem. However, XbaI and SpeI have compatible sticky ends which may result in uncontrolable orientation of the insertion as well as vector self-ligation (unless treated with alkaline phosphatase).
This year, we propose an alternative PCR protocol called 2-step PCR (there are only 2 steps each cycle) to solve this problem. This method is adapted from Takara PrimerSTAR Max DNA Polymerase product manual with slight alternation. The set-up is shown below:
For the reaction mixture:
DNA template: 200-300ng/50ul
Forward primer: 20pmol
Reverse primer: 20pmol
PrimeSTAR Max Premix（2×): 25ul
DdH2O: up to 50ul
For the reaction condition set-up:
Pre-denaturalization
98 degree celcius for 3minutes
Amplification cycles
95 degree celcius for 30 seconds
68 degree celcius for 60 seconds
Repeat for 30 cycles
Final elongation
72 degree celcius for 3 minutes
Using this method, we successfully added prefix and suffix to the bFGF gene and ligate the product to pSB1C3 backbone with ease.
Note that this method may yield unspecific amplification, thus agarose gel electrophoresis and gel extraction should be performed to obtain amplification products of right size. Moreover, additional experiments are required to determine the optimal parameters for this PCR reaction.
Results
For more details, please check out our [http://2017.igem.org/Team:SYSU-CHINA#/Demonstrate result page]!
In iGEM 2017, SYSU-CHINA constructed the circuit in which bFGF was fused to eGFP as well as 3xFLAG tags and expressed under the control of CMV promoter. Bone marrow stromal cells (BMSCs) were infected the with lentivirus containing the circuit and selected using puromycin. The transcription was measured using quantative PCR.
The transgenic BMSCs was cultured for 48 hours and the supernatant, theoratically containing certain growth factors, was collected. Scratch wound healing assays were performed using HEK293T cells with the collected supernatant. The data below suggested that bFGF (in concert with other soluable factors) produced by transgenic BMSCs is able to accelerate wound healing.
To further demonstrate the potentials of the bFGF-transgenic BMSCs, Collected supernatant mentioned above was used to culture uterine endometrium cells, and MTT assay were performed after 48 hours. The result indicated that bFGF secreted by engineered BMSCs is able to promote endometrium cell growth.
References
1 Yun, Y. R. et al. Fibroblast growth factors: biology, function, and application for tissue regeneration. Journal of tissue engineering 2010, 218142, doi:10.4061/2010/218142 (2010).
2 Nissen, N. N. et al. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. The American journal of pathology 152, 1445-1452 (1998).
3 Spyrou, G. E. & Naylor, I. L. The effect of basic fibroblast growth factor on scarring. British journal of plastic surgery 55, 275-282 (2002).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 409
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 347
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 165
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