Coding

Part:BBa_K2354011:Experience

Designed by: CHAN , WEI-JEN   Group: iGEM17_NTHU_Taiwan   (2017-10-21)


Applications of BBa_K2354011

We cloned the sequence of ER-alpha and Monobody into the vector, and then we transformed the plasmid into BL-21 competent cells, respectively. After transformation, we extracted both plasmids from the cells and the vector was validated by gel electrophoresis.

T--NTHU_Taiwan--Results--er_ecoli.png

E. coli with gene of ER-alpha



A successful cloning was verified from the results of a PCR performed with specifically designed primers according to the theoretically expected length of ER-alpha_INP fusion gene and Monobody (797bp).


T--NTHU_Taiwan--Results--er_mono_pcr.png

1 : RFP ( mRFP F-primer and mRFP R-primer ); 2 : IPTG (promoter) + INP + RFP2 (ER F-primer and ER R-primer ); 3 : IPTG (promoter) + INP + RFP 1 (ER F-primer and ER R-primer ) ; 4 : IPTG (promoter) + ER with red spot (ER F-primer and ER R-primer ); 5 : IPTG (promoter) + ER2 (ER F-primer and ER R-primer ); 6 : IPTG (promoter) + ER1 (ER F-primer and ER R-primer ); 7 : IPTG (promoter) + Monobody with red spot (Monobody F-primer and Monobody R-primer ); 8 : IPTG (promoter) + Monobody 2 (Monobody F-primer and Monobody R-primer ) ; 9 : IPTG (promoter) + Monobody 1 (Monobody F-primer and Monobody R-primer ); 10 : RFP ( VR primer and VF2 primer ); 11 : IPTG (promoter) + INP + RFP 2 ( VR primer and VF2 primer ); 12 : IPTG (promoter) + INP + RFP 1 ( VR primer and VF2 primer ); 13 : IPTG (promoter) + ER with red spot ( VR primer and VF2 primer ); 14 : IPTG (promoter) + ER2 ( VR primer and VF2 primer ); 15 : IPTG (promoter) + ER1 ( VR primer and VF2 primer ); 16 : IPTG (promoter) + Monobody with red spot ( VR primer and VF2 primer ); 17 : IPTG (promoter) + Monobody 2 ( VR primer and VF2 primer ); 18 : IPTG (promoter) + Monobody 1 ( VR primer and VF2 primer )


Function of ice nucleation protein (INP)



We compared the difference between E. coli expressed RFP and RFP-INP. After the lysis of culture expressing RFP-INP, most of RFP was on the fragments of the membrane. Due to the larger structure of plasma membrane, it is easily spin down. If RFP-INP is on the E. coli plasma membrane when we centrifuged the cell lysate, RFP-INP is spun down with the plasma membrane, and we see a red pellet.


On contract, after cell lysis of RFP expression E. coli, most of RFP suspended in the solution. Since the centrifugation force, we set in this experiment is not enough to spin down particles as small as RFP. We see RFP suspended in the supernatant even after centrifugation.


This experiment showed that INP can bring RFP to the membrane of E. coli, and this result proved the function of INP.

T--NTHU_Taiwan--Results--inp.png

1 : Fragments of E. coli with gene of RFP-INP ; 2: Fragments of E. coli with gene of RFP


To decrease the activity of E. coli, we tried to freeze E. coli in -80℃ for 24 hours and use it after thawing on ice immediately. We found that there is a significant difference between the sample with 5mM EDCs and EDCs-free and the intensity of IR signals also decrease due to the reduction of mobility of E. coli. This result can prove that our detection system can detect EDCs in the water.

T--NTHU_Taiwan--Results--ir2.png

The IR spectrum of samples in different conditions (freezed E. coli)



Proof of concept: quantifying a number of EDCs in the water



To prove our detection system can measure the concentration of EDCs, we use crystal violet to stain E. coli on the surface of gold and then observe the density of stained E. coli under microscope. Due to the surface tension force of water, there are lots of E. coli remained around the drop edge of sample, and we only observed the area in the middle of the sample to reduce the error.

T--NTHU_Taiwan--Results--demon.png

Tthe sample under 50X microscope


We observed the density of E. coli when the samples contained different concentration of BPA and NP from 5mM to 5nM. This result indicates that when the concentration of EDCs decreases, the density of E. coli will decrease as well.

T--NTHU_Taiwan--Results--bpa_500.png

The samples with different concentration of BPA under 500X microscope

T--NTHU_Taiwan--Results--np_500.png

The samples with different concentration of NP under 500X microscope


To ensure the function of ER-alpha on the surface of E. coli, we compared the difference between the BL-21 E. coli and ER-alpha expressed E. coli in the different concentration of BPA and NP. From the results, we found that BL-21 can’t affect the outcomes of the detection system.

T--NTHU_Taiwan--CBPA.png

Samples of BPA

T--NTHU_Taiwan--CNP.png

Sample of NP


User Reviews

UNIQ1327a28572e2a3b5-partinfo-00000000-QINU UNIQ1327a28572e2a3b5-partinfo-00000001-QINU