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Team:SYSU-China/file/Project/Notebook/Labnote/B2H.html
From 2014.igem.org
Contents |
B2H·LABNOTE
MAY
PLAN
pET-32 Plasmid extraction.
Test the competent cell.
Test Tet, Cmr antibiotic.
Try to use DH5α Amplify pBT and Ptrg, Then carry out the following work.
Continue to test the MRF competent cell.
Amplify the pTRG plasmid and send for sequencing.
Find a backbone in the Biobrick which can coexist with pBT and pTRG.
SUM UP
amplify pET-32 for reporter construction, but fail to obtain good quality plasmids.
prepare the XL1-Blue MRF competent cell for pBT and pTRG amplification but failed.
Conclusion this two things are not necessary! We were totally wasting our time! Still stuck on MRF.
Transformation of the MRF failed many times. We got very frustrated about the MRF.
Get functional Tet solution.
Get pBT plasmids (pBT-M and pBT-LGF). And do restriction enzyme analysis.
Transformation failed many times so we got the pTRG and pSB4A5 plasmid at the end of this week.
Get new Chloramphenicol and test its function.
Find out that maybe it was because the Tet agar plates have double antibiotic concentration.
Prepared competent cell ( DH5α).
JUNE
PLAN
Now we got all the plasmid backbone ready for molecular cloning work.
Construct pSB1C3-Or2 ( the promoter for B2H system). And then continue to construct the Reporter: pSB4A5-Or2-RBS-GFP.
Construct pBT-RFP and pTRG-RFP ( primers are ready and begin the PCR).
Obtain a sequence confirmed pSB1C3-Or2 plasmid.
Construct reporter plasmid by insert a fragment of RBS-GFP downstream of Or2.
Construct pBT-RFP and pTRG-RFP.
If we can obtain three plasmid, we can start trying three plasmid co-transformation.
Get sequence confirmed pBT-RFP and pTRG-RFP.
SUM UP
Test the new competent cells XYW made last week.
Transfer RBS+GFP from backbone pSB1A2 to pSB1C3 . ZJH failed once and XYW repeat that.
Test whether the concentration of three antibiotic is okay.
Construct the pSB1C3-Or2 plasmid. Do not catch up for sequencing on Friday afternoon because of colony PCR failed.
Obtain a sequence confirmed pSB1C3-Or2 plasmid.
Obtain pBT-RFP and pTRG-RFP single colonies and sent for sequencing
The pSB1C3-Or2 sequence result is wrong.
Get the right pBT-RFP and pTRG-RFP plasmid.
The 4th floor refrigeration house is FROZEN.
Got the sequencing result. We thought we insert a GFP downstream of Or2 but it turned out to be a YFP.
JULY
PLAN
Confirm the concentration of three antibiotics again ( co-transform on Monday)(done on Tuesday)
Prepare the plates for co-transformation.(at least 10)(by Tuesday afternoon)
Perform co-transformation ( Tuesday afternoon)
set 5 groups
premix all 5 groups for co-transformation
add plasmids to the competent cell ( BL21) revive for 45min?
spread on the 3-antibiotic plates
get single colony ( Wednesday morning)
Get single colony for the experiment groups (Wednesday morning)
Question: What would they look like? Do they look the same?
Induction (Wednesday)
Test the intensity of fluorescent
Construction of pLac-YFP and Or2-GFP and pLac-GFP
Construct plac-YFP plasmid
Finish construct pSB4A5-Or2-GFP plasmid and carry our three plasmid co-transformation
Add tag to GFP
SUM UP
Confirm the three antibiotic concentration and get photo of the experiment result.
Perform co-transformation of the B2H strains.
Meet some problem when culturing the strains in three antibiotic culture.
Finishing preparing the B2H strains.
Prepare positive control for fluorescent test (strain that can express RFP and YFP respectively)
Due to the plasmid of plac is somehow degredation, plac related experiment failed
pSB1C3-Or2-GFP is successfully constructed
Finish preparing competent cells
Get plac-YFP as positive control.
Get GFP co-transformation strains.
Decided it is not necessary to add tag to GFP.
AUGUST
PLAN
Design primers for mutants
Sequencing for pSB4A5-Or2
Check the C+ plate.
Test the inducement completely.
Go to TW for the meet up
Li Pai continue to repeat the B2H experiments
Prepare new BL21 competent cells ( the strain from 4th floor with lower leaky expression)
Confirm the colony of BL21 can be induced.
Continue to repeat the induction experiments
Decide whether it is necessary to use the reporter strain
Using BL21 from 4th floor to obtain co-transformation strains
See whether reporter cells can be infected by M13 phage? (how?)
SUM UP
Mutation of LGF2 and Inducement.
Solve the problem that the measuring of RFP expression strain using OD 600 is not that exactly.
Confirmed that the colony of BL21 can be induced.
Repeated the induction experiments
BL21 we prepared ourselves have leaky expression, but the origin doesn't
SEPTEMBER
PLAN
Use the confirmed strains to test fluorescents Draw the graph Plan for flow cytometry ( buy beats?)
Plan for part submission Design primers for λcI-LGF2 mutants Plasmid extraction of Or2?
Integrate the system. Design the strategy to integrate B2H system to other parts.
Finish Biobrick vector construction
Finish point mutation experiment and test fluorescent
Start doing western blot
B2H system integrated with RNAT
Test fluorescent of HML groups
SUM UP
Tested the fluorescents and draw the graph.
Integrated the system.
Constructed the Biobrick vector.
Finish point mutation experiment and test fluorescent
Western blot
B2H system integrated with RNAT
Fluorescent of HML groups tested.
OCTOBER
PLAN
Quick change for the λcI-LFG2
Test fluorescent of Or2-RNAT groups
Prepare for western of pSB4A5-Or2-p8
Try p3 again
Prepare for Gal11P-RNAα integrated into M13 plasmid
IPTG induction
P3 related strains spread the plate
Prepare to amplify the pBT-LGF2 plasmid
SUM UP
The 1000 group is significantly lower than 10 group
Test fluorescent of Or2-RNAT groups
For more imformation, you can click <a href="https://onedrive.live.com/redir?page=view&resid=DF8C73A38BBC1139!121&authkey=!ANYl53wdq2h4YA0" target="_blank">B2H NOTEBOOK</a>
