Difference between revisions of "Team:BIT-China/Notebook"

1. After discussion, the main goal of our project was to implement the self-killing of bacteria while the plasmid number reduces to a threshold.

1. Divided into three wet experiment group according to our project design. Killer group was to test the lethal efficiency of different killer genes. Inhibitor group was to test the threshold quantitatively. Integration group was to integrate the designed part into the genome.

1. Optimize the group division and prepare the some experimental reagents and equipment.

1. Decide to employ the CRISPR Cas9 coupled λ-Red recombination to improve the integration efficiency and simplify the selection process.
2. We got new parts pBAD (2015kit3 14A) andB0010 (2015 kit4 16G) from iGEM parts library. We transformed pBAD into TOP 10 and seeded/incubated in LB plates added chloramphenicol and transformed B0010 into TOP 10 and seeded/incubated in LB plates added Ampicillin.
3. This weekend，we designed the experiment of CRISPER/Cas9 team and made sure what circuits we need to construct.

1. Colony PCR：They had a positive result. And we incubated it in LB（Add antibiotics by 1000:1）.
2. We chosen TOP 10 as our host in this project, and looked for some genome information for preparation of recombination .
3. We chosen TOP 10 as our host in this project, and looked for some genome information for preparation of recombination .

1. And Send the plasmids for sequencing in order to confirm the parts. (pBAD,B0010)
2. Choosing lacZ as target gene, we designed the sgRNA of this was site through online tool.
3. Choosing lacZ as target gene, we designed the sgRNA of this site through online tool.

1. Design the primers of pBAD+B0034+LacIq
2. Through different online tool, we get different groups of sgRNA, after compare with each other, we chosen the best we thought.

1. The result of pBAD was positive，and B0010 was nagetive.

1. So We got new parts B0010 (2014 kit 4 16G) again, we decided to repeat transformation again.
2. We designed the primers and sent order to company, these primers will be used to construct the circuit EcoRI－N20-sgRNA－B0015-XbalI.

1. Colony PCR：Theyhad a positive result. And we incubated it in LB.

1． And Send the plasmids for sequencing in order to confirm the parts. (B0010)
2． We get the strains containing the sgRNA backbone plasmids, after bacilliculture, we extracted this plasmid. Plasmid PUC19 were transformed into TOP 10 and incubated in LB plates added Ampillin.

1. Run PCR for amplification of LacIq
2. Run PCR for amplification of LacIq
3. LacIq using pfu PCR program.
4. DNA clean/purification:
5. Purification of RCR/Restriction digestion production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.
6. Maked sure that PUC19 had been successfully transformed into TOP 10 vie colony PCR.
7. At the same time the another colony PCR result showed that the sgRNA was successfully ligated into plasmid PUC57.
8. Maked sure that PUC19 had been successfully transformed into TOP 10 vie colony PCR.

1. The result was negative.
2. To combine sgRNA and B0015 these two parts together, we run PCR for amplification of these two part separately, the result of agarose gel electrophoresis showed that sgRNA fragment was right but there was no B0015 fragment .

1. Designing the primers of B0010.
2. Purification of PCR production to get B0015 fragment, then run OE RCR to connect it with sgRNA fragment, so we get EcoRI－N20-sgRNA－B0015-XbalI this circuit.

1. We got plasmid contain λ-red gene and transformed it into TOP 10.
2. Purification of PCR production to get B0015 fragment, then run OE-PCR to connect it with sgRNA fragment, so we get EcoRI－N20-sgRNA－B0015-XbalI this circuit.
3. We get plasmid contain λ-red gene and transformed it into TOP 10.

1. Connect B0010 with primers，Purification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.

1. Connect pBAD+B0034+lacIq with primers,Purification of RCR production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.
2. We confirmed the positive colony of circuit EcoRI－N20-sgRNA－B0015-XbalI, after germiculture, these bacterial were sent to sequence.

1. Overlap-extension PCR to connect （pBAD+B0034+lacIq） with B0010.
2. Purification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.

1. Overlap-extension PCR to connect （pBAD+B0034+lacIq） with B0010.
2. Purification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.

1. Restriction digestion of gene circuit (pBAD+B0034+LacIq+B0010) with EcoRI and PstI at 37℃ for 4 hours.
2. Ligation of (pBAD+B0034+lacIq+B0010) with pSB1C3 by T4 ligase.

3. To design the left and right homologous arms, we needed exact sequence of lacZ gene. But in TOP 10, this gene was modified, so we design the primers contain EcoRI and PstI to get the lacZ gene, and got it sequence vie sequen
4. To design the left and right homologous arms, we need exact sequence of lacZ gene.But in TOP 10, this gene was modified, so we design the primers contain EcoRI and PstI to get the lacZ gene, and get it sequence vie sequencing.

1. Use Colony PCR to check whether the ligation worked as expected.
2. Colony PCR：They had a positive result. And we incubated it in LB.
3. Run PCR for amplification of lacZ gene, after purification, the fragment was digested by EcoRI and PstI, we also digested the vector PUC19 with same restriction enzyme.After 4 hours digestion, we ligated these two fragment.

1. And Send the plasmids for sequencing in order to confirm the parts. (pBAD+B0034+LacIq+B0010 and pTAC)
2. PCR was carried out on RFP and pTac using pfu PCR program.
3. Transformed the ligation produce of yesterday, and incubate the bacterial at 37℃.

1. Purification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.
2. The result was negative.
3. Transformed the ligation produce of yesterday, and incubate the bacterial at 37℃. The sequence result of 20th April showed that device 1 was right.
4. Run colony PCR to get the positive colony from yesterday’s plate. Incubate it and planed to sent it to sequence.

1. PCR was carried out on RFP and pTac using pfuPCR program again.
2. Purification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.
3. The result was positive.
4. Ligation of them by T4 ligase.
5. Run colony PCR to get the positive colony from yesterday’s plate. Incubate it and planned to send it to sequence.
6. Sent the bacterial to sequence.

1. Ligation products (pTAC and RFP) were transformed into TOP 10 and seeded in LB plates.
2. We decided to use mazF and HokD as our killer gene.

3. 3A assembly: pBAD-B0032-HokD, successful. pBAD+B0032+mazF, unsuccessful.

1. Designing the primers of which included B0034 to connect B0034 with pTAC+B0034+RFP.

1. We got the sequence result, just as we guessed, there were some difference in lacZ gene between TOP 10 and MG1655.

1. For the low quality of sequence, To confirm the sequence, we sent the bacterial to sequence again.

1. We called the company sold the BM TOP 10 for the exact sequence, but they rejected us.

1. Overlap-extension PCR to connect （pTAC+B0034） with RFP.
2. Purification of RCR production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.

1. Ligation of them by T4 ligase.
2. 3A assembly: pBAD-B0032-HokD, successful.
3. pBAD+B0032+mazF, unsuccessful.

1. Ligation products (pTAC+B0034+RFP) were transformed into TOP 10 and seeded in LB plates.
2. The sequence quality was bad still. We designed the primers again, plant to sequence it again.

1. Colony PCR：They had a negative result.
2. During this time, we got the exact sequence of lacZ, we designed the homologous arm referring to this sequence.
3. We began to constrict the Device3 and Device4.

1. Run PCR for amplification of pTAC and RFP. Purification of RCR production by running agarose gel electrophoresis.
2. we had positive result of pTAC and had negative result of RFP.
3. Extract the right brand pTAC by TIANgel Midi Purification Kit.

1. Run PCR for amplification of pTAC and RFP. Purification of RCR production by running agarose gel electrophoresis.
2. we had positive result of pTAC and had negative result of RFP.
3. Extract the right brand pTAC by TIANgel Midi Purification Kit.

1. Run PCR for amplification of pTAC and RFP. Purification of RCR production by running agarose gel electrophoresis.
2. we had positive result of pTAC and had negative result of RFP.
3. Extract the right brand pTAC by TIANgel Midi Purification Kit.

1. Ligation products (pBAD+B0034+LacIq+ B0015) were transformed into TOP 10 and seeded in LB plates.
2. We began to constrict the Device3 and Device4.
3. Device3: PstI－pBAD－B0034－λ_Red－B0015－BamH
4. Device4: BamHI－pBAD－B0034－Cas9－B0015－KpnI
5. These two devices would be constricted in vector pSTV29.

1. PBLB’s bacteria didn’t grew up.
2. Run PCR for amplification of pTAC-RFP again. Purification of RCR production by running agarose gel electrophoresis.
3. we had negative result of pTAC-RFP

1. Ligation products PBLB (pBAD+B0034+LacIq+ B0015) were transformed into TOP 10 and seeded in LB plates again.

1. Use Colony PCR to check whether the ligation worked as expected.
2. Colony PCR：We had a negative result.

1. Run PCR for amplification of PBLB (pBAD+B0034+lacIq+B0015) with Universal Primers

1. Use the TIANprep plasmid Kit to extract plasmids.
2. Send the plasmids for sequencing in order to confirm the parts.

1. We got the pSTV29 vector and transformed it into host DH5αto enrich it.

1. Run PCR for amplification of λ_Red gene and purify it.

1. Run OE PCR for connection of device 3 and device 4 separately.

1. We had a positive result. (PBLB)
2. Added the homologous arms to kana resistance gene, after digestion, it was ligated with pUC19 overnight.

1. Run PCR for amplification of PBR（PTAC+B0034+RFP）.
2. Transfer the ligation product into TOP 10, incubate it overnight.
3. Send the plasmids for sequencing in order to confirm the parts (RFP).

1. The concentration of sample was too low to test.
2. Run colony PCR and get the positive colony, after incubation, it would be sent to sequence.

1. Design the primers of pTAC+B0034 and add restriction site.

1. Run PCR for amplification of pTAC and RFP with primers.
2. Purification of RCR production by running agarose gel electrophoresis.
3. we had negative result of pTAC.
4. And Restriction digestion of RFP with XbaI and PstI.

1. Restriction digestion of pTac with EcoRI and SpeI
2. Restriction digestion of RFP with XbaI and PstI.
3. Ligation of pTac +RFP with pSB1K3 by 3A-assembly.
4. Device 3 fragment was get vie OE-PCR, after digestion, It was ligated with PSTV29 vector overnight.

1. Use Colony PCR to check whether the ligation worked as expected.
2. Colony PCR：We had a negative result.
3. Restriction digestion of pTac +RFP with EcoRI and SpeI
4. And we got negative result.
5. Transformed the ligation product into TOP 10, the result of colony PCR showed that there were no positive colony.

1. Run PCR for amplification of pTAC and RFP with primers.
2. Purification of RCR/Restriction digestion production by running agarose gel electrophores was. We had negative result of pTAC.
3. We checked the sequence of promoter and found the restriction site of enzyme we used, so we designed the promoter and changed the enzyme we used.

1. Run PCR for amplification of pTAC and RFP with primers again.
2. Purification of RCR production by running agarose gel electrophoresis.
3. Restriction digestionof pTAC with EcoRI and SpeI. We had negative result of pTAC.
4. We designed the primers to standardizing the device 1.
5. PCR: pTac+B0032+MazF, sessessful.
6. 3A assembly: pBAD+B0034+lacIq+B0015 and pTac+B0032+MazF with PSB1C3, and than, transformed it into TOP 10 and seeded in LB with chloramphenicol, unsuccessful.

1. During this time, we finished the standardization of device1. There were some problems still existed in construction of device 3 and device 4, we guessed that’s because these two devices were too long, and the length difference between these two devices was two large.

1. Run PCR for amplification of pTAC and RFP with primers again.
2. Purification of RCR/Restriction digest production by running agarose gel electrophoresis.
3. Restriction digestion of pTAC with EcoRI and SpeI.
4. we had positive result of pTAC.

1. 3A-assembly: Ligation of pTAC+RFP with pSB1K3 by 3A-assembly.

1. Ligation production pTAC+RFP were transformed into TOP 10 and seeded in LB plates

1. Use Colony PCR to check whether the ligation worked as expected.
2. Colony PCR: select the appropriate restructuring bacteria from 6.10 contained plasmids.
3. We found pBAD promoter always being closed without araC, so we decided replace pBAD to araC-pBAD.

1. Use the TIANprep plasmid Kit to extract plasmids.

1. Ligation products PBLB(pBAD+B0034+LacIq+B0015) were transformed into TOP 10 and seeded/incubated in LB plates again.

1. Ligation of pTAC and RFP with pSB1K3 by 3A-assembly.

1. Ligation products pTAC and RFP were transformed into TOP 10 and seeded/incubated in LB plates .

1. Use Colony PCR to check whether the ligation worked as expected.
2. Colony PCR: select the appropriate restructuring bacteria from 6.24 contained plasmids.

1. Use the TIANprep plasmid Kit to extract plasmids.
2. Send the plasmids for sequencing in order to confirm the parts.

1. We designed the primers of device4’s constriction, planned to divided the cas9 gene into two fragment and constrict it by two steps.

1. We finished the constriction of first plasmid and added the araC gene in front of pBAD promoter. The constriction of device4 was finished.

1. There were something wrong still exited in constriction of device 3, we wanted to constricted it by Gibson assembly.
2. OE: araC-pBAD-B0034 + LacIq-B0015

1. check the sequence of device 2, make sure that there were no recognition sites of EcoR1、XbaI、SpeI、PstI these four enzyme. The result from DNAman showed that no recognition site should been mutated
2. design the primers which contained the four enzymes’ recognition sites, the primers were showed below;

3. we used these two primers clone device2,by this way, after purification and recovery, we got the device2 fragment with prefix and surfix.
4. Cut both modified device2 fragment and pSB1C3 with EcoR1 and PstI;
5. Ligate device2 fragment and pSB1C3 with ligase;
6. Transfer the last step product into competent cell TOP 10;
7. Spread the last step product on the LB plate contain chl, 37℃ overnight incubated ；
8. Check the single colony via COLONY PCR , and sequence the positive one.

9. transferred araC-pBAD-B0032-HokD into cells

10. araC-pBAD-B0032-HokD colony PCR, 4&5positive result, picked up the thallus and preserve recovered araC-pBAD-B0032-MazF, unsuccessful

11. araC-pBAD-B0032-HokD distracted plasmids, genetic sequencing

12. Ran colony PCR of araC-pBAD-B0032-MazF fragment

1. We selected the constructed bacteria through COLONY PCR . But the result was all negative.
2. In order to connect the gene circuit araC-pBad-B0034-LacIq-pTac-B0032-RFP through GOLDEN GATE assembly, we added restricted sites to three parts, araC-pBad, B0034-LacIq-B0015, pTac-B0032-RFP, respectively through OE-PCR. And we transformed these three parts into E.coil strain TOP 10, via three vectors P O T.
3. (gram-positive bacteria on July 12th) araC-pBAD-B0032-MazF distracted plasmids, genetic sequencing

1. In order to test the efficiency of RFP, we added IPTG into our LB medium when bacteria’s OD had arrived 0.6. But the experimental group was the same as group control. The result elucidated that the RFP can not work.
2. GOLDEN GATE ASSEMBLY : use restriction enzyme BsaI to splice these three parts we got on 7.13. And integrate into P O T plasmids. Transform into E.coli .
3. PCR recovery:

1. Extract the plasmid: extract the plasmid from the bacteria tested on 7.14. And send for DNA sequencing.
2. GOLDEN GATE ASSEMBLY: select the bacteria via COLONY PCR and inoculate it into LB medium.
3. OE-PCR: connect the gene circuit araC-pBad-B0034-LacIq-B0015-pTac-B0032-RFP, the gel result was positive!
4. Digested/Ligated/Transformed:

1. Transformation: get the part K864400 (pTac) from iGEM kit library for exchanging the promoter before.
2. Transformation: integrate the gene circuit made on 7.15 into plasmid pSB1C3 by restriction digestion and ligation. Then transform it into E.coli strain TOP 10.
3. GOLEDEN GATE ASSEMBLY: Build the gene circuit araC-pBad-B0034-LacIq-B0015-pTac-B0032-RFP and integrate it into the plasmid, POT, by restriction enzyme BsmBI. Then transform the constructed plasmid into E.coli strain TOP 10.

1. GOLDEN GATE ASSEMBLY: select the bacteria through COLONY PCR . But the result was negative. Because the gene LacIq contains the BsmBI recognition sites.
2. OE-PCR: the transformation performed on 7. 16 was failing. We had to connect the gene circuit araC-pBad-B0034-LacIq-B0015-pTac-B0032-RFP again.
3. ONE-STEP MUTATION: mutate the B0032 into B0034 by PCR in order to improving the expression of RFP for testing easily.
4. Inoculated the bacteria contained pTac(K864400).

1. We design three pairs of primers to clone two 500bp homologous arms from TOP 10 genome and the kana resistance gene from device 2;
2. After recovering the product of last step, we linked them one by one via OE PCR, and we recovered the product of this step.
3. We got Pkd46 from doctor Bo Lv and transferred it into TOP 10.
4. Incubate TOP 10 get from last step, induce TOP 10 express Beta, Exo, Gam(three key proteins of this system) by L-arabinose(10mmol/L)
5. Transfer the donor DNA into TOP 10 via electroporation, 37℃ incubated 1hour,during this time, add IPTG to induce expression of kana resistance gene;
6. Spread the product on the plate contain kana,
7. After 16 hours’ incubation, there were some colonies on the plate. But the result of colony PCR showed that there was no positive result.
There were some factors maybe impact the result:

8. EXTRACT THE PLASMID: Extract the plasmid contained the pTac from the bacteria.
9. ONE-STEP MUTATION: Nitrification the PCR product by DpnI.

1. OE- PCR: Build the new gene circuit pTac-B0032-RFP.
2. Get parts cI (C0051) and pR (R0051) from the iGEM Kit library. And transform into E.coli strain TOP 10
3. Determined growth curve in host cells(the antibiotic in controlled plates might have lost efficiency)：

1. EXTRACT THE PLASMID: get the plasmid contained the parts cI and pR .
2. COLONY PCR : Test the pTac-B0032-RFP by COLONY PCR . But the result was negative.
3. Digested/Degradation/Transformed：

1. OE-PCR: Connect the gene circuit araC-pBad-B0034-cI-b0015-pR-B0032-RFP.
2. Inoculate the constructed bacteria contained gene circuit pTac-B0032-RFP.
3. Ran colony PCR:

1. OE-PCR: Connect the gene circuit araC-pBad-B0034-cI-B0015-pR-B0032-RFP.
2. Inoculate the constructed bacteria contained gene circuit pTac-B0032-RFP.

1. EXTRACT THE PLASMID: Extract the plasmid from bacteria inoculated on 7.23. And the gel result was positive. And we sended the sample for sequencing.
2. OE-PCR: Construct the new gene circuit araC-pBad-B0034-cI-B0015-pR-B0032-RFP.
3. So we changed the primers and carried out the plan again. There were better result of PCR, but no positive result still. So we decided to give up lacZ as target gene.

1. 3A ASSEMBLY: Connect the araC-pBad-B0034-cI-B0015-pR-B0032-RFP by 3A assembly. And we also build the circuit by OE-PCR at the same time.
2. Get the part tetR(C0040) and pTet-B0034-RFP-B0015(I13521) from the iGEM kit library. And transform these plasmids into TOP 10 for accumulating plasmid.

1. 3A ASSEMBLY: Connect the araC-pBad-B0034-cI-B0015-pR-B0032-RFP by 3A assembly.
2. Inoculate the bacteria contained the tetR and pTet-B0034-RFP-B0015 for accumulation.
3. Determined growth curve in host cells

1. EXTRACT THE PLASMID: Extract the plasmid contained tetR and pTet-B0034-RFP-B0015.

2. Designing the primers of PBTB（pBAD+B0034+tetR+B0015）
3. SDS page
4. Designing the primers ofPBTB（pBAD+B0034+tetR+B0015）

1. We got new parts tetR and（pTet+B0034+RFP+B0015）from iGEM parts library. We transformed them into TOP 10 and seeded/incubated in LB plates .
2. 3A ASSEMBLY: The bacteria which only had the gene circuit pR-B0032-RFP did not express the RFP. And we extracted the plasmid for the further connection. But the result of the restriction digestion about araC-pBad-B0034-cI-b0015 was incorrect. The restriction site SpeI was wrong.

3. The sequencing about araC-pBad-B0034-LacIq-B0015 was incorrect. So we had to build this gene circuit again. And we built the gene circuit araC-pBad-B0034-tetR-B0015.
4. SDS page

5. We got new parts tetR and（pTet+B0034+RFP+B0015）from iGEM parts library. We transformedtheminto TOP 10 and seeded/incubated in LB plates .

1. Use Colony PCR to check whether them worked as expected. Incubate the positive bacteria in LB medium.
2. SDS page
3. Use Colony PCR to check whether themworked as expected.

1. we cloned the left arm and right arm from genome of TOP 10, cloned the MRFP Expression Cassettes from plasmid tested by the Inhibitor group. After recover these three fragment, we assemble them vie OEPCR, at last, we recovered the product of OEPCR;
2. the TOP 10 contain Pkd46 were prepared in the same time;
3. Transfer the donor DNA into TOP 10 vie electroporation method.
4. Spread the product of last step on blank plate and plate contain ampicillin, incubate them in 37℃ overnight.

5. Use the TIANprep plasmid Kit to extract plasmids.
6. Construct the the gene circuits

1. Prime Star :

2. Prime Star :

③araC pBAD+tetR+B0015

1. PCR:

2. Ligation of araC+ pBAD+tetR+B0015 and RFP with pTet+RFP by 3A-assembly.
3. urification of RCR/Restriction digest production by running agarose gel electrophoresis. Extract the right brand by TIANgel Midi Purification Kit.

1. PCR:

2. Use Colony PCR to check whether the ligation worked as expected

3. We build the gene circuits:

4. We then decided to select the recon by antibiotic resistance. So we added a Chloramphenicol resistance Expression Cassettes into donor DNA, so after recombination, the right recon will have Chloramphenicol resistance and red look.But before we carry out a new plan, we plan to check competent cell, and make sure pkd46 was transferred into it. We extract plasmid from competent, the result of electrophoresis showed that no pkd46 in it, we decided to use new competent cell.

1. recovery：

2. Use the TIANGEN plasmid Kit to extract plasmids. Send the plasmids for sequencing in order to confirm the parts.

1. Ran primer star PCR:

1. Got GFP plasmids from kit box, transformation

1. Transformation on August 7th, unsuccessful

2. Contains PKD3 plasmids and PKD46 plasmids bacteria. Transfer into liquid culture medium. Put it in the shaking table for 12 hours. Preserve the bacteria then extract PKD3 plasmids and PKD46 plasmids.PKD46 provide theλ-red gene and PKD3 provide the chl gene.

1. repeated OE&PCR

2. GFP distracted plasmids, unsuccessful

3. Ran primer star PCR/Recovery:

4. Ran pfu PCR/recovery/Ran OE PCR: B0032+GFP with new template (sequenced) unsuccessful
5. Get Amp Plate out of incubator. Use colony PCR find the positive results. But we fail.

6. Use colony PCR find the positive results .And we fail again.

1. In the TOP 10 plate ,there no bacteria grow up, but in the DH5αplate ,there were some single colonies. Transfer it into liquid culture medium and induce the promoter with 10%Ara.

2. Until now, we have one contrast.DH5αcell and TOP 10 cell. In order to analysis how many homologous arms were more appropriate, we design two homologous arms, one was 555bp and another was 49bp. We have two kinds of aim snippets. Now, we have two contrasts.

1. Ran OE-PCR with different systems and annealing temperatures, successful

2. PCR/Recovery: B0032-GFP

3. Recovery, unsuccessful

4. Troubleshooted, primers’ error

5. Ran PCR: araC-pBAD-B0032&GFP

6. The result was that too many colonies in the A and C plate, but none bacteria grow up in the D plate. The bacteria only grow up in the B plate. By the verification of colony PCR , the result doesn’t have any positive bacteria.

1. We use the mRFP upstream and downstream primers, after electrophoresis we get the wrong stripe again.
2. So we decide to check the whole snippet. We use VF and VR to copy the whole snippet, the whole snippet was about 2053bp, but the snippet we get was about 300bp , that was clearly wrong.

1. Now we were confused. Why always can't successfully transfer into the TOP 10 cell? Why did it grow up in the B plate but doesn’t have positive results?
2. We thought about the above wassues, the only way to make it have Chl gene was transfer the PKD3 plasmids into the cell by mistake. In order to get the Chl gene, we use PCR technology to get it from PKD3 plasmids. So we make a verification experiment.
3. We extract the plasmid from B plate, use it to be the complete for cat gene, and we succeed. And we also do electrophoresis to compare with PKD3 plasmid, we get the same stripe. It shows that the Chl gene was brought by PKD3. To solve this problem, we nitrify after we copy the whole snippet. It will eliminate plasmids.

1. We copy the Chl gene snippet and mRFP gene snippet by PCR technology. After electrophoresis we get the right stripe then we use kit recycle the snippet and the last we verify the snippet.
2. Use OE-PCR link two snippets.

1. Copy the whole snippet through PCR technology, after electrophoresis we get the wrong stripe.
2. Use the bacteria we preserved at August 8. Transfer into liquid culture medium. Make it competent cell.

1. Copy the whole snippet through PCR technology, after electrophoresis we get the right stripe. Then we use kit recovery the snippet and verify the snippet. Nitrify the whole snippet for two hours. But the product concentration was too low
2. Design a 49bp new homologous arm for Chl gene.
3. Because we purpose fragment replacement LacI locus, we use VF and VR to copy the lacI snippet, after electrophoresis we get the right stripe. It told us that our experimental thinking was correct, so we can replace this locus.
4. The product concentration was too low. We use PCR technology again to enrich the snippet. We use the already nitrification fragments as templates so we don't need to nitration after PCR.

1. Use electrophoresis to verify the whole snippet, but it was not correct.
2. Use OE-PCR connects two snippets again. This time we made it. After electrophoresis we get the right stripe then we use kit recovery the snippet and the last we verify the snippet. The result was correct.
3. Use electric turn to transfer it and lay over in the Chl plate.

1. We inspect the Chl plate, there was no single colony.
2. Do the paper work and prepare for tomorrow’s experiment.
3. Culture it in the 30℃ incubator overnight.

1. Before we make competent cell, we use 1mL 10%Ara induceλ-red Reorganization system for 2 hours, when we make competent cell, we make two kind of concentrations, one was diluted 10 times, another was diluted 100 times.
2. Recover for one hour.
3. Lay over the Chl plate. Culture it in the 30℃ incubator overnight.

1. Use colony PCR find the positive results.
2. After electrophoresis we get negative results except one have two snippets, one was 1000bp and another was 2000bp.Transfer this bacteria into liquid culture medium. Mark①.
3. There were some new single colonies in the Chl plate, so we use colony PCR find the positive results again.

4. We pick these single colonies and transfer into liquid culture medium to see if it lose Chl resistance. If so, the aim snippet did not link to DH5α genome.

5. We measured the concentration:

1. Observe bacteria①under Fluorescence microscope. No red fluorescence.
2. Observe bacteria①under Fluorescence microscope. No red fluorescence.
3. Use colony PCR find the positive results.

4. Used pfu PCR program run PCR:

1. Connected the purpose fragment to T carrier. Transfer into DH5αcell. Lay over DH5αcells in the CHL plate.
2. Though Colony PCR to texting. The result was right. Transfer it into liquid culture medium.
3. Sent to sequencing.
4. Ran colony PCR to validate the transform result: araC-pBAD-B0034-CI-B0015-Pr-B0032-MazF -PSB1C3, unsuccessful.
5. PCR was carried out on araC-pBAD-B0034 and B0032-GFP using pfu PCR program, unsuccessful.
6. We ran PCR for amplification of pTac-B0032-MazF and pR-B0032-MazF, successful.
7. We cultured bacteria which contain araC-pBAD-B0032-HokD into LB media with chloramphenicol overnight.
8. Kept the bacteria which contain araC-pBAD-B0032-HokD and abstracted its plasmid, successful.
9. We recovered the gelatin which contains araC-pBAD-B0034 and B0032-GFP, low concentration.
10. We cultured bacteria which contain araC-pBAD-B0032-MazF into LB media overnight.
11. Kept the bacteria which contain araC-pBAD-B0032-MazF and extracted its plasmid, successful.
12. we ran two overlap-extension PCR to connect pTac-B0032/pR-B0032 with MazF, successful.

1. .Observe bacteria under Fluorescence microscope. No red fluorescence.

1. Observe TA cloning bacteria under Fluorescence microscope. It has red fluorescence.
2. Restriction digest of pTac-B0032- MazF and pTet--B0032- MazF with XbaI and PstI, at 37°C for three hours. And then put them into 80°C water bath for 20min to stop the reactions.
3. Ligated of araC-pBAD-B0034-CI-B0015 (reverse consequence) and Pr-B0032-MazF with PSB1C3 by T4 ligase.
4. araC-pBAD-B0034-CI-B0015 (reverse consequence), pR-B0032-MazF and PSB1C3 were transformed into TOP 10 and seeded in LB with chloramphenicol, unsuccessful.
5. Ligation by T4 ligase overnight:
6. araC-pBAD-B0034-LacIq-B0015(reverse consequence) and pTac-B0032-MazF with PSB1C3; araC-pBAD-B0034-TetR-B0015(reverse consequence) and pTet-B0032- MazF with PSB1C3.

1. Help other group. We have one circuit.
2. Restriction digest:
3. pR-B0032 with MazF, and then put them into 80°C water bath for 20min to stop the reactions.
4. 3A assembly:

5. Transformed into TOP 10 and seeded in LB with chloramphenicol:

1. Design new primers for three circuits.

2. colony PCR :

3. Transformed into TOP 10 and seeded in LB with chloramphenicol:

1. PCR and recovered the gelatin: pR-B0032-MazF,

2. colony PCR :

3. Extracted plasmid:

1. Get these three fragments through PCR technology.
2. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct.
3.Culture glycerin bacteria(DH5α) into liquid culture medium.
4. Restriction digest:

5. 3A assembly:

6. Sequenced:

7. We cultured bacteria which contain PSB1C3 into LB media overnight.

1. Through OE PCR to link two fragments. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct.

6. colony PCR :

7. We cultured bacteria which contain araC-pBAD-B0034-TetR-B0015(reverse consequence)-pTet-B0032- MazF-PSB1C3 into LB media overnight.
8. Ligation by T4 ligase overnight:

9. araC-pBAD-B0034-CI-B0015(reverse consequence) and pR-B0032-MazF with PSB1C3.

1. Use colony PCR to find the positive result in crossed bacteria (TA colony)。
2. After electrophoresis we get the right stripe. Last time we text this fragment, it showed that the bacteria was not single clone. We pick other bacteria and text them, to make sure we have single colony.
3. Add TA colony bacteria into liquid culture medium.
4. Extract plasmids and run the plasmids, the result have two stripes. So we extract genome.
5. Make competent cell. Transfer these three fragments

6. They were spread on LB agar plates with CHL on at 37℃.
7. Transformed into TOP 10 and seeded in LB with chloramphenicol:

8. Kept the bacteria which contain :araC-pBAD-B0034-TetR-B0015(reverseconsequence)-pTet-B0032- MazF-PSB1C3 and extracted its plasmid, successful.
9. PCR:

1. Bacteria have grown up so we use colony PCR to find the positive results.
2. We use different primers and no positive results.
3. colony PCR :

4. Sequenced: araC-pBAD-B0034-TetR-B0015(reverse consequence)-pTet-B0032- MazF-PSB1C3
5. Cultured bacteria into LB media overnight with chloramphenicol:

6. 3A assembly:

7. Restriction digest and recovered the gelatin:

1. Use colony PCR to find the positive results again. Results: no positive results.

2. Digest the fragments.
3. Use OE PCR to connect two fragments. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct.
4. Sequenced:

5. 3A assembly:

6. First round of PCR: For amplification of mutated promoters at 65°/67° with upstream primers JB1, JB2, JB3. [Pfu protocol]

1. The liquid culture medium has been contaminated. We can’t transfer our bacteria. We make up culture medium and sterilize.
2. When the culture medium was ready. We transfer our bacteria and culture it overnight.
3. olony PCR :

araC-pBAD-B0034-LacIq-B0015(reverse consequence)-pTac-B0032-MazF-PSB1C3;
araC-pBAD-B0034-CI-B0015(reverse consequence)-pR-B0032-MazF-PSB1C3,
successful.

4. Cultured bacteria into LB media overnight with chloramphenicol :

1. Culture the bacteria into another culture medium for 2.5 hours.
2. Make the competent cell. Transfer our fragments into competent cell.
3. They were spread on LB agar plates with CHL on at 37℃.
4. The sequencing results came out. Almost right except some base mutated.
5. Culture TA colony bacteria into liquid culture medium.
6. Explanation: The bacterium was transferred to the inhibitor group for texting.
7. Restriction digest:

8. PCR for amplification:

9. Replace the PCR machine/the template, and do it again.

1. Use colony PCR to find the positive results.

2. Make TA colony bacteria into competent cell. The plasmid was constructing. But the cell concentration was too low. We will do it again next day.
3. Add TA colony bacteria and DH5αcell into liquid culture medium.
4. 3A assembly:

5. PCR was carried out on [pTet+B0034+RFP+B0015] circuit using Fast Pfu Fly Program. Extension time was set to 50s and annealing temperature was 67°.

1. Culture them again. Use Arabinose to induce DH5αbacteria.
2. Make TA colony bacteria and DH5αbacteria into competent cell.
3. Put TA colony bacteria competent cell in the -80℃.
4. Transfer our fragments into competent cell.
5. They were spread on LB agar plates with CHL on at 37℃.
6. colony PCR :

7. Cultured bacteria into LB media overnight with chloramphenicol:

8. Restriction digestion of JP1/JP2/JP3 with EcoRI and PstIat 37℃ for 2h20min. Purification of Restriction digest production by running agarose gel electrophoresis. Extract the right the right brand by TIANgel Midi Purification Kit.
9. Restriction digestion of pSB1C3 with EcoRI and PstIat 37℃ for 2h20min. urification of Restriction digest production by running agarose gel electrophoresis. Extract the right the right brand by TIANgel Midi Purification Kit.
10. Ligation of JP1/JP2/JP3 with pSB1C3 by T4 ligase.
11. Ligation products JP1/JP2/JP3 were transformed into TOP 10 and incubated in LB plates added Chlorine.

1. Use colony PCR to find the positive result.
2. Enrich fragments to improve concentration. After electrophoresis we don’t get the stripe.

3. Culture DH5αcell into liquid culture medium.
4. Make DH5αcell into competent cell.
5. Transfer our three fragments into DH5α competent cell.
6. They were spread on LB agar plates with CHL on at 37℃.
7. Culture it overnight.
8. 11 Measured growth curve(induced and uninduced)：

9. We Used Colony PCR to check whether the ligation worked as expected. For amplification of mutated promoters at 55℃ with Single colony.

10. Send the plasmids for sequencing in order to confirm the parts.

1. Use colony PCR to find the positive result. And we succeed.
2. The positive results were sent to sequencing.
3. Cultured bacteria into LB media overnight with chloramphenicol:

4． Colony PCR: select the appropriate restructuring bacteria from Sept 11. contained plasmids. Only
5． araC-pBad-CI-B0015-PR-B0032-RFP-B0015 and araC-pBad--B0034-Laciq-B0015-pLac-b0032-YFP-B0015 had positive results. And incubate NO.24(laciq) and NO.32(CI) in LB with kanamycin.

1. Measured growth curve(induced and uninduced)：

2. Ligation products (araC-pBad-tetR-B0015-Ptet-B0032-RFP-B0015, J23119-B0032-CI-B0015,

3. Extract the plasmids:

4. Incubate araC-pBad-CI-B0015-PR-B0032-RFP-B0015 and araC-pBad--B0034-Laciq-B0015-pLac-B0032-YFP-B0015 into LB follow the form:

1. Get the PCP20 plasmid. Next step was to transfer this plasmid into the fragment that we already sequencing.
2. Use the TIANprep plasmid Kit to extract plasmid then we verify the plasmid. It was correct.
3. Design the experiment to eliminate the CHL resistance and the PCP20 plasmids.
4. Transer the DHαcell what including our aim fragment and PKD46 plasmid in the culture medium at 37℃ for 2 hours and 42℃ overnight. In order to eliminate the PKD46 plasmid, reduce the burden of bacteria.
5. The plates did not grow except J23119-B0034-Laciq-B0015(only one).
6. OE-PCR: connect the araC-pBad with tetR-B0015 again. fast PFU fly program. And the PCR results were positive. And we purified the PCR production by TIANgel Midi Purification Kit.

7. Group A and group B incubated last night did not show any differences incubated last night. The same result as C and D. But the control, group E incubated pSB1C3, did not grow and there were some deposition in LB medium.

1. Streak of single colony.
2. Use the TIANprep plasmid Kit to extract plasmid. Run PCR amplification of this plasmid. The plasmid was already eliminated.
3. Continue verification.
4. We tested the promoter strength.

5. Test: araC-pBad-CI-B0015-PR-B0032-RFP-B0015 (reverse)
6. Colony PCR: araC-pBad-tetR-B0015 had a positive result. And we incubated it in LB.

1. Use colony PCR to verify the PKD46 plasmid.

2. Culture the bacteria in LB medium

3. first round of PCR: For amplification of mutated promoters at 67℃ with upstream primers SJTB. [Pfu protocol]

4. Target Fragment observed through Gel electrophoresis.

4. Purification with kit from TIANGEN Biotech. 5. OE-PCR:

6. Digestion:

1. Make competent cell and transfer the PCP20 plasmid.
2. Incubate the bacteria in LB medium with AMP.
3. Culture it overnight.
4. Measured growth curve(induced and uninduced)：

5. Colony PCR:

6. Digestion:

1. The concentration of the competent cell was too high, so the bacteria grow all the AMP plates.
2. We do it again and culture it overnight.
3. We use Fluorescence microscope to observe the bacteria and find they all emit red fluorescence. But two of them supposed to emit yellow fluorescence. We decide to limit the gene circuit again.

4. Colony PCR:

5. Test the araC-pBad-CI-B0015-PR-B0032-RFP-B0015 again. But we found our sample was polluted by other bacteria. So we tested the arabinose (only add arabinose into LB ).
6. We got new parts J23109, J23116 and J23106 from iGEM parts library. And transform them into TOP 10. And we also got new vector pSB3K3, pSB4K4 at the same time.

1. Use OE PCR to link three fragments with CHL resistance gene.
2. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct.
3. Make competent cell and transfer the fragments.
4. Incubate the bacteria in LB medium with CHL.
5. Culture it overnight.
6. Digestion and ligation:

7. We Used Colony PCR to check whether the ligation worked as expected. For amplification of mutated promoters at 55℃ with Single colony.

1. Test the araC-pBad-RFP-B0015 to simulate the inhibitor.
2. PCR:

1. Transformation:

1. Colony PCR:
The results of transformation made on 9.20 were negative.

1. Make competent cell. Transfer these three fragments (PR-B0034-Yfp-B0010+B0012+cat

1. No bacteria grow in the LB culture medium
2. The sequence results were wrong. We decided to do it again.
3. Us OE PCR to enrich the fragments.
4. Culture the DH5αand TOP 10 bacteria in the LB culture medium.
5. araC-pBAD-B0034-CI-B0015 (reverse consequence) and pR-B0032-MazF with PSB1C3, successful.
Send the plasmids for sequencing in order to confirm the parts.

1. Restriction digestion of plac+Yfp,PR+mRFP,PR+Yfp and Ptet+mRFP with EcoⅠand SpeⅠat 37℃ for 3 hours.
2. Then we use kit recovery the snippets and the last, we verify the snippet.
3. Ligate of plac+Yfp,PR+mRFP,PR+Yfp and Ptet+mRFP with PSB1C3 by T4 ligase. Transfer it in the TOP 10 cell.
4. Measured growth curve(induced and uninduced)：
araC-pBAD-B0034-HokD;
PSB1C3.
5. Test the araC-pBad-CI-B0015-PR-B0032-RFP-B0015 again for confirming the system’s function. (9.24-9.25)
6. Colony PCR (9.24):

7. OE-PCR: restructure the gene circuit araC-pBad-tetR-B0015.(9.25-9.26)

1. Use colony PCR to find the positive results. Succeed.
2. This was inhibitor team’s circuit.

3. PCR was carried out on this circuit using Primer Star PCR program. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct.
4. Transfer it into TOP 10 cell.
5. Not succeed.
6. 3A assembly: araC-pBAD-B0034-lacIq-B0015(reverse consequence) and pTac-B0032-MazF with PSB1C3,
unsuccessful.
7. For amplification of mutated promoters at 67℃ with upstream primers SJTB . Reduce the annealing temperature. [Pfu protocol]
Target Fragment observed through Gel electrophoresis.

8. Purification with kit from TIANGEN Biotech.
9. Digestion and ligation:
Restriction digestion of SJTB with EcoRI and PstIat 37℃ for 2h20min. Purification of Restriction digest production by running agarose gel electrophoresis. Extract the right the right brand by TIANgel Midi Purification Kit.
10. Ligation of SJTB with pSB1C3 by T4 ligase.
11. Ligation products SJTB were transformed into TOP 10 and seeded/incubated in LB plates added Cholrine.
12. Transformation :

1. Restricition digestion of J23119-B0032/34-tetR/CⅠ-B0015 with ECORⅠ and PSTⅠat 37℃ for 6 hours.
2. Use kit to recovery it.
3. Ligation of J23119-B0032/34-tetR/CⅠ-B0015 with PSB1K3 by T4 ligase.
4. Transfer it into TOP 10 cell. Incubate the bacteria in LB medium with Kan.
5. Not succeed.
DO it again.
Not succeed.
6. Measured growth curve(induced and uninduced)：
araC-pBAD-B0034-HokD;
PSB1C3,.
7. 3A assembly:

1. We got these four fragments from PSB1K3 by restriction digestion, using EcoⅠand SpeⅠ.

2. Restricition digestion of J23119-B0032-tetR-B0015,J23119-B0034-tetR-B0015,J23119-B0032-CI-B0015 and J23119-B0034-CⅠ-B0015 with ECORⅠ and PSTⅠat 37℃ for 6 hours.
3. Use kit to recovery it.
4. Ligation of J23119-B0032-tetR-B0015,J23119-B0034-tetR-B0015,J23119-B0032-cⅠ-B0015 and J23119-B0034-CI-B0015 with PSB1K3,PSB3K3 and PSB4k5 by T4 ligase.
5. Transfer them into TOP 10 and incubated in LB plates add KAN.
7. Use colony PCR to verify it. Correct.
8. Send to sequence.
9. Restriction digestion of SJTB with EcoRI and PstIat 37℃ for 2h20min. Purification of Restriction digest production by running agarose gel electrophoresis. Extract the right the right brand by TIANgel Midi Purification Kit.

10. Test the gene circuit:

11. Construction:

1. Make competent cell and transfer J23119-B0032-tetR-B0015 and J23119-B0034-tetR-B0015 into DH5αcell which contain Ptet-B0032-mRFP-B0015 fragment, transfer J23119-B0032-CⅠ-B0015 and J23119-B0034-CⅠ-B0015 into DH5αcell which contain PR-B0032-mRFP-B0015 fragment.
2. There were too many bacteria grow in the KAN plate, so we make plate streaking.
3. Measured growth curve(induced and uninduced)：

4. We Used Colony PCR to check whether the ligation worked as expected. For amplification of mutated promoters at 55℃ with Single colony.

5. Send the plasmids for sequencing in order to confirm the parts.
6. Colony PCR:

7. Colony PCR:

1. Use colony PCR to verify it. Not succeed.
2. We culture our competent cell in KAN plate, it grow up. It was strange, because our competent cell don’t have KAN resistance. We culture other glycerol bacteria in KAN plate, in order to see if they have KAN resistance.
3. Test the gene circuit araC-pBad-RFP-B0015.

1. The glycerol bacteria grow in the KAN plate, we need to change the host cell and do it over again.
2. PCR was carried out on plac+Yfp+cat,PR+mRFP+cat,PR+Yfp+cat and Ptet+mRFP+cat using Primer Star PCR program. These fragment was use to recombine.

3. After electrophoresis we get the right stripe then we use kit recovery the snippets and the last, we verify the snippet. The result was correct
4. Restriction digestion of plac+Yfp+cat,PR+mRFP+cat,PR+Yfp+cat ,Ptet+mRFP+cat with DPNⅠat 37℃ for 4 hours. Restriction digestion of plac+Yfp+cat,PR+mRFP+cat,PR+Yfp+cat ,Ptet+mRFP+cat with EcoRⅠand PstⅠat 37℃ for 4 hours.
5. Use kit to recovery all the fragments.
6. Ligation of biobricks with PSB1C3 by T4 ligase.
7. Transfer PKD46 plasmids into DH5αcompetent cell.
8. Use colony PCR to verify it.

9. SDS-PAGE: test the Jilin University’s device.
10. Digestion:

11. Ligation(standardization):

1. Transfer the products of ligation into TOP 10 and incubated in LB plates add CHL.
2. Incubate the DH5α in LB medium with CHL. Culture it overnight.
3. Fluorescence measurement:

3. Standardization:

4. Colony PCR:

1. Incubate in a new LB medium.
2. Make competent cell and transfer PR+mRFP+cat, Ptet+mRFP+cat into competent cell.
3. Incubated in LB plates add CHL.
4. Incubate the DH5α in LB medium with CHL. Culture it overnight.
5. For amplification of mutated promoters at 67℃ with upstream primers SJTB.

6. Purification with kit from TIANGEN Biotech.

7. PCR:

8. Colony PCR:

1. There was no bacteria grow in the CHL plates.
2. Incubate in a new LB medium.
3. Make competent cell and transfer PR+mRFP+cat, Ptet+mRFP+cat into competent cell.
4. Incubated in LB plates add CHL.
5. Incubate the DH5α in LB medium with CHL. Culture it overnight.
6. Fluorescence measurement again:

7. Colony PCR:

1. There was no bacteria grow in the CHL plates.
2. Incubate in a new LB medium.
3. Make competent cell and transfer PR+mRFP+cat, Ptet+mRFP+cat into competent cell.
4. Incubated in LB plates add CHL.
5. Construction:

1. There was no bacteria grow in the CHL plates.
2. Ligation of J23119-B0032/B0034-CⅠ/tetR+B0015 with PSB1C3 by T4 ligase.
3. Transfer them into TOP 10 and incubate in LB medium with CHL.
4. Standardization:

5. Colony PCR:

1. Digestion:

2.Colony PCR: