Team:SCUT/Notebook
Notebook
Introduction
This year our project aims to dealing with heavy metal ion--Cd2+,considering about the amount of work, we divided our project into two parts: intracellular Cd2+concentration sensing signal circuit structure and extracellular surface adsorption. As shown in figure, the protein MerR can repress PcadA with Cd2+ absence, which makes downstream gene TetRcan’t express so that it can’t repress Ptet. As a result, fluorescincjbluegets expression. In another word, in low concentration, E.coli’s color is blue-green. While in high concentration, Cd2+ can enter E.coli by MntA’s active transport, leading to intracellular Cd2+ concentration increase. At this time, MerR’s repression is weakened, RFP gets expression. At the same time, cjblue stop expressing with tetR’s expression so thatE.coli becomes red gradually.
Cd2+ concentration signal sensing
1.MerR construction lab notes
Members in this charge of this part: Zhong Yu, Cai Xiao Jun, Liu Sen Biao.
Introduction: We built the pathway for Cd2+ ion to enter into the E.coli ,we had constructed parts as follow:
PcadA+MerR+ MntA+ ter
PJ23100+MerR+ MntA+ ter
PJ23101+MerR+ MntA+ ter
PJ23106+MerR+ MntA+ ter
PcadA+MntA+ ter
PJ23100+MntA+ ter
PJ23101+MntA+ ter
PJ23106+MntA+ ter
2015.5-2015.7
Contents: PCR techniques, transformation of plasmid,constructed PcadA 、PJ23100、PJ23101、PJ23106 on PSB1C3.
5.1-5.15
Content: Obtained PcadA 、PJ23100、PJ23101、PJ23106 by overlap PCR
1.Digested Vector and PcadA 、PJ23100、PJ23101、PJ23106
2.Assembled PcadA 、PJ23100、PJ23101、PJ23106 to the pSB1C3 with pSB1C3 on it.
3.Verification
Notes:We failed for the first time because of the false recycling.
5.20-6.30
Contents: Moloecular construction
Note:we had some trouble on the way to get PcadA 、PJ23100、PJ23101、PJ23106 through PCR techniques, we can not obtain target gene from plasmid by PCR directly because these promoter were too short, so we adopted overlap PCR to get them.Later then,we successfully constructed :
PcadA+MntA+ ter
PJ23100+MntA+ ter
PJ23101+MntA+ ter
PJ23106+MntA+ ter
for modeling group to measure related parameter.
Similarly,we adopted the same method to obtain:
PcadA+MerR+ MntA+ ter
PJ23100+MerR+ MntA+ ter
PJ23101+MerR+ MntA+ ter
PJ23106+MerR+ MntA+ ter
to connected with the part of “Cd2+ adsorption” for integration expression.
Conclusion: we managed this part of moloecular construction smoothly without too much problems,which made our experiment in an orderly manner. Having finished this part ,we had time to give assistance to our partner , cooperation makes our team more united ,and we gained a lot of joy at the same time.
2.Concentration signal sensing
Members in this charge of this part: Lin Xiao Mei, Luo Xun xun, Cui Tian Hua.
Introduction: This part moloecular construction as follow:
Ptet-cjblue-ter
Without the Cd2+ existence , Ptet didn’t resist the expression of cjblue ,and we can see blue fluorescence in bacterial colony.In this way we can distinguish the expression difference of E.coli under the condition of Cd2+ existence or not.
2015.5-2015-6
Contents: Obtained Ptet and cjblue through PCR techniques,we digested Ptet and cjblue,they were assembled in plasmid pSB1c3.
Notes: We found some problems at the beginning,we can’t get the Ptet due to the false we made in primer designing,we re-designed the primer and got the right gene successfully.Besides, during the recovery of PCR product,No plastic recycling caused empty PSB1C3,later then we change the way of recycling,this trouble was dealt smoothly.
Conclusion: Moloecular construction of this part was relatively simple as last part.Although we met some problems ,we corrected them in time to make our plan developed,which was worth for us to feel happy,and it made us believe in ourselves that we could finish the project perfectly at the same time.
Extracellular surface adsorption
moloecular construction:
PcadA+CsgA-EC0+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC1+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC2+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC3+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC5+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
May
Week 1
We got the Top10 strain to prepared for our experiment and we used restriction enzyme to digest single gene (tetR、RFP)on pSB1c3,and made a ligation with terminator.
Week2
We successfully constructed tetR-RFP-ter on pSB1c3,and next we prepared our gene for PcadA-CsgA-EC（0、1、2、3、5）.
Week3
We started to do the PCR in order to get our targeted gene PcadA and CsgA-EC，we got the CsgA（EC0）from company，so EC would be link with CsgA during the PCR procession，EC（2、3、5）had to adopt two-round PCR due to their length.
Week4
We obtained PcadA and CsgA ,we tried to link them,however,we found no colony on culture medium. Although we reviewed the literature for a longtime and repeated our operation ,the problem still wasn’t be overcome.
June
During the month,we were trying to find out the reason why CsgA can’t fit PcadA successfully.We checked the correctness of gene,even we restarted from the first step,we gained nothing.
July
Week1
Benefited by mutual discussion,we adopted overlap PCR to obtain PcadA-CsgAEC(0、1、2、3、5) directly. Primers were designed, and we waited for their coming.
Week2
With the PcadA designed on the primer,we successfully constructed PcadA on CsgA-EC（0、1、2、3、5）
Week3
The PcadA-CsgA-EC(0、1、2、3、5）were digested by restriction enzyme ,then they were loaded on the pSB1c3 which contain the gene of tetR-RFP-ter.
Week4
We successfully constructed
PcadA+CsgA-EC0+tetR+RFP+ter
PcadA+CsgA-EC1+tetR+RFP+ter
PcadA+CsgA-EC2+tetR+RFP+ter
PcadA+CsgA-EC3+tetR+RFP+ter
PcadA+CsgA-EC5+tetR+RFP+ter
During this part we met many kinds of problems so that we had spent many time but we can not improve the efficiency,which meant we had to reflect ourselves.
August
Week1
We planed to finish our work this month,we digested PcadA+CsgA-EC+tetR+RFP+ter with restriction enzyme and linked with Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter and Ptet+ CJBlue+ter+PJ23100+MerR+ ter respectively.
Week2
After we finished transformation and inoculated ,we sent our plasmids for sequencing,however ,nothing was found on pSB1c3.
Week3
We repeated the transformation but found that the colony we transformed last week turned blue,which meant false positive colony grows faster than target colony.
Week4
We adopt inoculation to blue colony and sequencing.The result show that the sequence of target genes were correct.
September
Up to now,all moloecular construction had been finished.We successfully constructed
PcadA+CsgA-EC0+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC1+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC2+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC3+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
PcadA+CsgA-EC5+tetR+RFP+ter+Ptet+ CJBlue+ter+PJ23100+MerR+ MntA+ ter
Having finished moloecular construction we sped up composing our wiki,On the other hand we started to prepare posters and the presentation at the same time and detected parameters by modeling group to perfect our project.
Parts obtain
moloecular construction
Test the device
