Difference between revisions of "Part:BBa K3332079"

Revision as of 16:55, 27 October 2020

pBAD/araC-Inverter-EYFP-terminator

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

Usage and Biology

Fig 1. pBAD_inverter_eyfp_B0015

This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity/OD600 of induced and non-induced bacteria, we managed to characterize the function of the inverter.

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. 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 (about 3000bp)

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:

Fig 3. Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

We found that the downstream gene of proBAD/araC can be expressed when induced by arabinose, while the expression of EYFP decreases without the inducer. Besides, as we can see, the purple bar is higher on the right side(when the arabinose is absent). Therefore, we can come to the conclusion that when the inverter is added, 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 20: / Line 20: @@
 ===Characterization===
-When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used the ''EcoR'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
+When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used the ''Eco''R 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]]
-'''Fig.2''' pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by ''EcoR'' I and ''Pst'' I (about 3000bp)
+'''Fig.2''' pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by ''Eco''R I and ''Pst'' I (about 3000bp)
 '''Protocol: '''
@@ Line 54: / Line 54: @@
 '''Fig 3.''' Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
-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.
+We found that the downstream gene of proBAD/araC can be expressed when induced by arabinose, while the expression of EYFP decreases without the inducer. Besides, as we can see, the purple bar is higher on the right side(when the arabinose is absent). Therefore, we can come to the conclusion that when the inverter is added, the effect is reversed.
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