Fig1. The diagram of conception and basic design
As our mainly idea of the specificly selectivity gene circuit apporched by synthestic biology, whetehr the cell division initiated and selection marker exist are the most impotaint characteristics. (A) Schematics of a AND gate between two sensors. (B) An
illustration of actral setup in DNA seqence. hBax-β, an example of output. (C)Selectivity of circuit in different cell line.
A
B
C
Fig.2 The flow chat with fusing 4 repeats miR-142 microRNA binding site.
The sequnces of miR-142 are seperaed into two pairs of pre-annealed oligonucleotides
which contain one sticky end and one blunt end of restrcition enzyme at P1 and P4 sequences (BglII and EcoRV). By using the T4 PNK to transfer the γ-phosphate from ATP to the 5’-OH group, miR-142 MBS can ligate with vector to create a new recombi-nation vector.
Fig.3 The aligements of procaspase-3 and cleaved caspase-3 ; procasepase-7 and
truncated caspase-7.
To accelerate the protein maturation, the prodomain of both caspase-3 and caspase-7 is cleavaged initialy. Morever, we chose the truncated c-caspase-7 mutated at amino acid 57, is reported that triggers apoptosis frequenly than other mutant type. (A) The procaspase-3 gene is cloned form whole genome in the DH5α strain. Compared with the DNA sequence reported online, we construct the activated caspase -3 by removed the
prodomain. (B) To construct the truncated caspase-7, the prodomain is removed first.
Refer to the supporting information from previous study, amino acids 57-303 of caspase-7 shows the morphologies of apoptosis after activated and causes the significant cell A
B
death than others mutant. Hence, we design the primers which can bind on caspase-7 sequence started form amino acid 57 to remove the amino acid 1-56.
A
B
Fig.4 The expression level of reporter triggered by full length (949) or truncated
(342) cyclin B1 promoters at G0 and G2 phase
The first sensor is chosen because the abnormal division and unlimited proliferation are the most common feature in cancer cells. We sythcrozined the transfected HeLa cells by double thymidine assay and relased for 2 hours that allows cells to go into G2 phase. As the control group, we also block cells into G0 phase, let cells have no ability to activate the cell cycle by cultured in certain medium which is lack of nutrients for 72 hours.
Obseved the fluorescence by microscope, the reporter (mCherry) triggered by (A) truncated cyclin B1 promoter (342 CBP) and (B) full length cyclin B1 promoter (949CBP) shows significantly high expression in G2 phase than G0 phase. (C) The percentage of fluorescent-positive cells indicate that the 342 CBP can successfully
regulate the gene accroding the cell cycle.
C
B A
HeLa cells 293T cells
Fig.5 The transfected cancer cells which constitutively express the toxic gene results
in cell apoptosis in HeLa cells and 293T cells.
(A) The illustration of constructs containing different toxic gene and with or without miR-142-5p MBS located at the downstream triggered by CMV promoter. The EGFP reporter is used as transfected maker. (B) The image of cell morphology result that the overexpression of hBax-β gene and truncated caspase-7 gene can efficiently killing the cancer cells. The transfected cells expressing cleavage caspase-3 gene
show the EGFP and caused less cell death after transfected for 24 hours, which means that overexpression the cleavage caspase-3 alone doesn’t have the enough ability of killing the cancer cells. We also co-transfect the two toxic genes to know whether the killing ability is more powerful than expressed alone or not. In this re-sults, the cleavage caspase-3 and truncated caspase-7 shows the significant killing
co-work with hBax-β gene.
Fig.6 The inducible promoter triggers the toxic gene results in cell apoptosis.
The circuit is containing the tetOn-3G system regulated by 342 CBP and toxic gene activated by TRE promoter. If the Dox exist, the toxic proteins are produced and cause the cell death. This result explains the same conclusion of the data mention above (Fig.5B). We consider the hBax-β gene shows the most powerful killing than others and successfully induced the apoptosis happened. Control: transfected 342CBP_TetOn +
mCherry and pTRE_EGFP. Positive control: transfected 342CBP_TetOn + mCherry and pTRE_EGFP (treated with STLC for 24 hours)
Fig.7 Time lapse image of HeLa cells transfected pTRE3G-BI_ hBax-β+EGFP after
inducing for 24 hours by doxycycline.
As this result, the HeLa cells start producing the hBax-β and EGFP in just 30 minutes after inducing by Dox. After 24 hours, most positive-transfected cells round up and
blebbing that means that cells are going to apoptosis.
Fig.8 Measurement the cell viability and cell death rate by trypan blue exclusion
assay in HeLa cells.
To measure the cell survival rate or death rate , we stain the transfected cells with try-pan blue and calculate the living cells and death cell by Hemocytometer. All the cells are co-transfected with 342CBP-TetOn + mCherry and toxic gene derived by
pTRE3G-BI.( A) Cell viability. (B) cell death rate. This data shows that most of the living cells number are decreased except the pTRE_EFGFP + c-caspase3::miR-142-5p MBS. pTRE_EGFP indicate the survival cells and pTRE_EGFP(STLC) indicate the cell death control. ns, non-siginificant(P > 0.05);**,P ≤ 0.01;***, P ≤ 0.001;**** P ≤
0.0001 (P values)
A B
(%)
Fig.9 Flow chat of cloning the construct containing constitutive and inducible
pro-moter.
In order to make a vector that can continuously produce the reporter gene and activate the toxic gen only when we add the inducer, we combinate the pBI-CMV1 and
pTRE3G-BI by digested with XhoI and XbaI. There are one side consist of CMV
en-hancer and CMV promoter and another side consist of 7X tet operator and TRE prom-ter.
Fig.10 Designs of vector and two promoters and insulator.
(A) Illustration of 2x core element of insulator located between two different promoters.
Moreover, we anticipate the cell would change the color form red to yellow after doxycycline exist, which express the red and green fluorescent at the same time. (B) The design of constructing the vector containing the insulator. We use the SalI to
ligate with XhoI since they have the same cutting site. According to this design, there A
B
is a scar happened but it doesn’t effect next enzyme digest and we can inset another core element of insulator as much as we want.
A
B
Fig.11 The blocking ability of different copies of core elements of insulator in HeLa
cells.
There are three kind of copies between two of the promoters (A) no insulator (B) 1x core insulator (C) 2x core insulator are used as our test group. No matter the inducer
exist or not, the mCherry driven by CMV promoter is produced all the time in all the groups. The EGFP driven by TRE promoter is significantly decreased in insulator-tained group than no insulator group. As this result, we can simplify learn that this con-struct can actually block the leakage between two different type of promoters.
C
Fig.12 Quantify the expression level of reporter triggered by two promoters
con-taining different copies insulator.
(A) The construct (Fig.11A) and Tet-On system controlled by 342 CBP are co-trans-fected into HeLa cells. As the result, the expression level of mCherry triggered by
CMV promoter has no change after adding the doxycycline, which means the con-stitutive promoter can stably express the downstream gene. (B) Expression level of EGFP is significantly decreased that containing one or two copies core element of
insulator. (C) Adding the doxycycline is attended, EGFP can be up to the same ex-pression level as the one without insulator. As the conclusion, the problem of leak-age can be resolved by inserted the insulator between two promoters. (n=20) ns,
non-siginificant(P > 0.05); **** P ≤ 0.0001 (P values)