Part:BBa_K1926002

A cyclic promoter of Ki-67 from human genome

Promoter of Ki-67
BioBrick No.	BBa_K1926002
RFC standard	RFC 10
Requirement	pSB1C3
Source	Addgene
Submitted by	[http://2016.igem.org/Team:SYSU-CHINA SYSU-CHINA 2016]

Ki-67 is a cell proliferation marker which is tightly associated with cell division[1]. In detail, except for G0 phase, the Ki-67 protein is present in all phases of the cell cycle ( including G1, S, G2 and M)[1]. Owing to the fact that Ki-67 gene only transcribe once in every cell cycle, the Ki-67 promoter must lead to transcription of the downstream DNA sequence once in every G1 phase.

According to Pei, D.S., et al., whom first cloned the 5’-flanking region of the human Ki-67 gene and located the Ki-67 core promoter, the Ki-67 promoter is the TATA-less, GC-rich region comprised of several putative Sp1 binding sites, one human zinc finger 5 protein (ZF5) consensus element, and one cell-cycle gene homology region (CHR)[1].

As for the function of Ki-67 promoter, it has been proved that it had higher transcription activity compared with the hTERT promoter and Survivin promoter[1].

For its regulation, it has been proved that the binding side of transcription factor Sp1, a ubiquitous transcription factor, existed in the Ki-67 core promoter. Besides, this promoter was proved to be repressed by interferon regulatory factor 1 (IRF1) in human Ketr-3 and 786-O renal carcinoma cells [2] and inhibited by p53 via p53- and Sp1-dependent pathway [3].

Design Considerations

The sequence was retrieved from Addgene. We got it from human genome through PCR using the following primers (details in Figure1):

pKi67-F: ACCTCTGCCCTCCGCCAGCCG

pKi67-R: ACCCGGTGGCCCTACAGGCTACG

Figure 1: The detail of primers designed for pKi-67, including its length, Tm, GC%, self-complementarity, and product length.

Owing to the fact that Ki-67 promoter is a eukaryotic promoter, eukaryotic vector is needed to insert it into cell genome. The tet-on vector we chose to use for our project, which comes from our host lab, had Sph1 and Xho1 restriction sites. With the two sites we can cut off the primary PGK promoter of the vector. Therefore, we add Sph1 and Xho1 sequence to the primers in figure1, digest PCR product with enzyme Sph1 and Xho1 and insert the digested parts into the tet-on vector which also digested by Sph1 and Xho1 in order to replace the PGK promoter with the Ki-67 promoter (Figure 2).

Figure 2: The map of the primary tet-on vector (a) and Ki-67 promoter in tet-on vector (b). The selected portion of the map shows that the sequence between Sph1 and Xho1 was changed from PGK promoter to Ki-67 promoter.

A better way to construct biobricks shorter than 1kb

When we need to construct biobricks, the first thing we need to do is to add the prefix and suffix to our parts. However, both prefix and suffix have 22bp in length with high GC%, which is unfriendly for PCR because the matching region of the primer with the part should be no shorter than the unmatching tail. As a result, the primers would be no shorter than 44bp in total length, accompanied by a huge Tm. If the GC% of the parts matching region is also high (such as the part is in a promoter in this case), the situation will become even worse. To address this problem, we decided to Add XbaI at the 5’ region of the part and SpeI at the 3’ region by PCR with a pair of “short” primers (Promoter-Fs and -Rs). For control, we also design a pair of “long” ones (Promoter-Fl and -Rl).

Promoter-Fs: 5’-TTCTAGAG - CAGTTTGGACTAGCATTCTA -3’

Promoter-Rs: 5’-TACTAGTA - ATATCATTTTACGTTTCTCG-3’

Promoter-Fl: 5’-GAATTCGCGGCCGCTTCTAGAG - CAGTTTGGACTAGCATTCTA -3’

Promoter-Rl: 5’- CTGCAGCGGCCGCTACTAGTA - ATATCATTTTACGTTTCTCG -3'

(before the ‘-’ is the unmatching tail while after the ‘-’ is the matching region)

With the “short” primers, we successfully construct two biobricks in this part collection (all G1 cyclic promoters, from Part:BBa_K1926001 to Part:BBa_K1926003) through the following protocol:

1. Add XbaI at the 5’ region of the part and SpeI at the 3’ region by PCR.

2. Digest pSB1C3 and the PCR product with XbaI and SpeI enzyme. Follow the instruction of the enzyme protocol.

3. Treat the digested pSB1C3 with alkaline phosphatase, which can remove the phosphate group of the cohesive ends of the backbone. Follow the instruction of the enzyme protocol.

4. Ligate the digested part and the digested-treated backbone, and transform into E.coli.

5. Product confirmation: After plasmid extraction, use enzyme XbaI digest the plasmid, of use Xbal and SpeI to digest the plasmid.

(See details and explanations of this protocol here [http://2016.igem.org/Team:SYSU-CHINA/Measurement/InnovativeBioBrickConstruction SYSU-CHINA 2016 Measurement])

For the “long” primers, we failed to use the Takara PrimerSTAR Max DNA Polymerase to get PCR products. When we change to use the expensive and tardy KOD enzyme in our host lab, we finally succeeded in having bands on our AGE image. Therefore, although with the “long” primers we can also construct biobricks, we think that for parts shorter than 1kb, our new protocol with the “short” primers is a better and cheaper choice.

Gel analysis after EcoR1 digestion

The length of our biobricks are confirmed by gel analysis after EcoR1 digestion. The biobrick BBa_R0040, which is the negative control in 2016’s interlab, was used as a control because it only have a 54 bp part.

Figure 3: AGE image of biobricks BBa_K1926001-BBa_K1926003. The samples were pre dyed with gelred.

Promoter function confirmation

G1 promoter function confirmation by transient transfection using 293T cells. Photos taken 48 hours after transient transfection, 10x. pCDK4, pKi67 and pCCNE are our G1 promoters. pmPGK is the constitutive promoter of mouse PGK, it is a medium promoter, here used as a control.

Figure 1: in vivo testing pG1 promoter function in human 293 cell line.

Use this part in your project!

You may use this part to:

1) Express something in mammal cell lines particularly in G1 phases or once in every cell cycle by stable transfecting it into cell line;

2) Use it as a human promoter by transient transfecting it into cells.

Reference

1. Pei, D.S., et al., Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription. Tumour Biol, 2012. 33(1): p. 257-66.

2. Chen, F., et al., IRF1 suppresses Ki-67 promoter activity through interfering with Sp1 activation. Tumor Biology, 2012. 33(6): p. 2217-2225.

3. Wang, M.-J., et al., p53 regulates Ki-67 promoter activity through p53- and Sp1-dependent manner in HeLa cells. Tumor Biology, 2011. 32(5): p. 905.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 425
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 422