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From 2011.igem.org

Project I.

1.1：The construction of pSB1AC3（BBa_K603000）/PR

 

 

 

 

 

 

 

 

PR + pSB1AC3 = pSB1AC3/PR (new part)

Cut PR w/EcoRI & SpeI

Cut pSB1AC3 w/EcoRI & SpeI

Combine the insert and vector with T4 ligase

1.2: Gel electrophoresis

1: wide range maker 2、3、4、5：the constructed plasmid cutted with SpeI and EcoRI restriction enzymes

 

 

 

 

 

 

 

 

 

 

1.3: DNA sequencing

The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.

 

2.1 The construction of pSG5/PR which is used for eukaryotic expression

PR + pSG5 = pSG5/PR
Cut PR w/EcoRI & BamH I
Cut pSG5 w/EcoRI & BamH I
Combine the insert and vector with T4 ligase

2.2 Gel electrophoresis

1: wide range marker

2: the constructed plasmid pSG5/PR cut with EcoR1 and BamH1

2.3 DNA sequencing

The result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

3.1  The construction of pBABE/PR

1) PCR for amplifying more insert
Primer 1:         5'-ggg gaa ttc taa acc acc atg ctt gcc-3'
Primer 2:         5'-aaa gaa ttc tca cta tta ggc gtt gct-3'
Reaction system:
10XPCR buffer      5ul
dNTP               4ul
primer 1           1ul
primer 2           2ul
pfu                1ul
template           1ul
DMSO               5ul
ddH2O              31ul
Total              50ul
Reaction condition:
95℃       5min

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95℃       15s
52℃       15s
72℃       2min

25cycles

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72℃       10min
4℃        ∞

2) Ligation

PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase

3.2 Gel electrophoresis

1:Middle range maker 　　　 1:DL2,000 DNA Marker

2:cut with EcoR I　　　　　 2:cut with Sal I

Figure 1 indicates that the insert is combined with the vector successfully.
Figure 2 indicates that the insert is in the right direction.

3.3 DNA sequencing

As before, the result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

 

 

4.1 The construction of BBa_K603001

fig.1 Proteorhodopsin(PR)digested at EcoRI and PstI and ligased to pSB1C3(Linearized plasmid backbone Pre-cut at EcoRI and PstI ends).

4.2 Gel electrophoresis

 

fig.2 In this fig,NO.1 is Wide range DNA marker,NO.2-4 are plasmid BBa_K603001 digested at EcoRI and PstI,demonstrate that the plasmid is correct.

Project II

1.The construction of lacI-DBD-LXR-LBD

fig.1 Amino acid sequence of lacI-DBD-LXR-LBD chimeric protein,the DNA binding domain of lacI is boxed.

The DNA sequencing(bottom left)and the gel electrophoresis of the digested pET28(bottom right) both demostrate that

the construction of lacI-DBD-LXR-LBD is exactly what we designed before.

2.The gene worknet

3.The experiment

fig.1 The gene circuit of the two-plasmid system.

fig.2 The key element that affect the RFP are IPTG,Kan and our compound.

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fig.3 We have tried five compounds,compound B,C,D and E can active the gene and express rfp.The pET28a is

control that have no lacI-DBD-LXR-LBD gene.

Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.