Difference between revisions of "Part:BBa K3332079"

Revision as of 04:28, 25 October 2020

pBAD/araC-Inverter-EYFP-terminator

It can express EYFP without arabinose and express little EYFP induced by arabinose.

Usage and Biology

This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity of induced and non-induced to characterize the function of the inverter.

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the EcoR I and Pst I to cut the plasmid, then we got the target separate fragment.

Fig.2 pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by EcoR I and Pst I

Protocol:

1. Preparation of stock solution

Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 2 mL arabinose stock solution into the induction group when OD600 increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Here is the result:

We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1205
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3332079 short</partinfo>
-It can express cyan fluorescent protein according to the concentration of arabinose.It is used to characterize the function of the inverter.
+It can express EYFP without arabinose and express little EYFP induced by arabinose.
 ===Usage and Biology===
-This part was used to detect and test formaldehyde promoter expression in BL21 as well as the promoter improvement design. After formaldehyde induction, ECFP would emit fluorescence under 514 nm exciting light and indicate the strength and degree of formaldehyde promoter expression.
 [[File:T-XMU-BBa K3332079.circuit.png|none|500px|caption]]
+This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity of induced and non-induced to characterize the function of the inverter.
 ===Characterization===
-When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the ''EcoR'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment-2476bp
+When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the ''EcoR'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
 [[File:T-XMU-BBa K3332079.ger.png|none|500px|caption]]
-Fig2. [BBa_K3332079] I0500_Q04510_E0430_pSB1C3 (pBAD_inverter_E0430_pSB1C3) digested by ''EcoR'' I and ''Pst'' I
+Fig.2 pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by ''EcoR'' I and ''Pst'' I
+Protocol:
-Protocol:
+. Preparation of stock solution
-. Preparation of HCHO stock solution
+Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）
 .Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
-.Add 4ml of the above bacterial solution into 100 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
+.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
-.Add HCHO stock solution into the induction group（the working concentration is 0.8mM） when OD increased to 0.6. The culture condition is the same as before.
+.Add 2 mL arabinose stock solution into the induction group when OD600 increased to 0.6.
-.Then, sampling 0.5ml culture in each tube. All samples are centrifuged at 12000rpm, 1 minute. Remove supernatant and add 500µl sterile PBS to resuspend.
+.Induce for 6 hours and the condition is the same as before.
-.Measure the fluorescence intensity(ECFP)and corresponding OD600  by 96-well plate reader, then calculate the fluorescence / OD 600 value of each group.
+.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
 Here is the result:
+[[File:T--XMU-2082.fig.3.dem.png|none|500px|caption]]
+We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed.
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