Team:SYSU-China/file/Project/Notebook/Labnote/B2H.html

From 2014.igem.org

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<h1>B2H·LABNOTE</h1>
<h1>B2H·LABNOTE</h1>
<!--SYSUCHINA!-->
<!--SYSUCHINA!-->
 +
<h2><b>MAY</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>pET-32 Plasmid extraction. </p>
 +
<p>Test the competent cell. </p>
 +
<p>Test Tet, Cmr antibiotic. </p>
 +
<p>Try to use DH5α Amplify pBT and Ptrg, Then carry out the following work. </p>
 +
<p>Continue to test the MRF competent cell. </p>
 +
<p>Amplify the pTRG plasmid and send for sequencing. </p>
 +
<p>Find a backbone in the Biobrick which can coexist with pBT and pTRG. </p>
 +
 +
 +
 +
<p><b>SUM UP</b></p>
 +
<p>amplify pET-32 for reporter construction, but fail to obtain good quality plasmids. </p>
 +
<p>prepare the XL1-Blue MRF competent cell for pBT and pTRG amplification but failed. </p>
 +
<p>Conclusion this two things are not necessary! We were totally wasting our time! Still stuck on MRF. </p>
 +
<p>Transformation of the MRF failed many times. We got very frustrated about the MRF. </p>
 +
<p>Get functional Tet solution. </p>
 +
<p>Get pBT plasmids (pBT-M and pBT-LGF). And do restriction enzyme analysis. </p>
 +
<p>Transformation failed many times so we got the pTRG and pSB4A5 plasmid at the end of this week. </p>
 +
<p>Get new Chloramphenicol and test its function. </p>
 +
<p>Find out that maybe it was because the Tet agar plates have double antibiotic concentration. </p>
 +
<p>Prepared competent cell ( DH5α). </p>
 +
 +
 +
 +
<h2><b>JUNE</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>Now we got all the plasmid backbone ready for molecular cloning work. </p>
 +
<p>Construct pSB1C3-Or2 ( the promoter for B2H system). And then continue to construct  the Reporter: pSB4A5-Or2-RBS-GFP. </p>
 +
<p>Construct pBT-RFP and pTRG-RFP ( primers are ready and begin the PCR). </p>
 +
<p>Obtain a sequence confirmed pSB1C3-Or2 plasmid. </p>
 +
<p>Construct reporter plasmid by insert a fragment of RBS-GFP downstream of Or2. </p>
 +
<p>Construct pBT-RFP and pTRG-RFP. </p>
 +
<p>If we can obtain three plasmid, we can start trying three plasmid co-transformation. </p>
 +
<p>Get sequence confirmed pBT-RFP and pTRG-RFP. </p>
 +
 +
 +
 +
 +
<p><b>SUM UP </b></p>
 +
<p>Test the new competent cells XYW made last week. </p>
 +
<p>Transfer RBS+GFP from backbone pSB1A2 to pSB1C3 . ZJH failed once and XYW repeat that. </p>
 +
<p>Test whether the concentration of three antibiotic is okay. </p>
 +
<p>Construct the pSB1C3-Or2 plasmid. Do not catch up for sequencing on Friday afternoon because of colony PCR failed. </p>
 +
<p>Obtain a sequence confirmed pSB1C3-Or2 plasmid. </p>
 +
<p>Obtain pBT-RFP and pTRG-RFP single colonies and sent for sequencing</p>
 +
<p>The pSB1C3-Or2 sequence result is wrong. </p>
 +
<p>Get the right pBT-RFP and pTRG-RFP plasmid. </p>
 +
<p>The 4th floor refrigeration house is FROZEN. </p>
 +
<p>Got the sequencing result. We thought we insert a GFP downstream of Or2 but it turned out to be a YFP. </p>
 +
 +
 +
 +
<h2><b>JULY</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>Confirm the concentration of three antibiotics again ( co-transform on Monday)(done on Tuesday) </p>
 +
<p>Prepare the plates for co-transformation.(at least 10)(by Tuesday afternoon) </p>
 +
<p>Perform co-transformation ( Tuesday afternoon) </p>
 +
<p>set 5 groups </p>
 +
<p>premix all 5 groups for co-transformation</p>
 +
<p>add plasmids to the competent cell ( BL21) revive for 45min? </p>
 +
<p>spread on the 3-antibiotic plates</p>
 +
<p>get single colony ( Wednesday morning) </p>
 +
<p>Get single colony for the experiment groups (Wednesday morning) </p>
 +
<p>Question: What would they look like? Do they look the same? </p>
 +
<p>Induction (Wednesday) </p>
 +
<p>Test the intensity of fluorescent </p>
 +
<p>Construction of pLac-YFP and Or2-GFP and pLac-GFP</p>
 +
<p>Construct plac-YFP plasmid</p>
 +
<p>Finish construct pSB4A5-Or2-GFP plasmid and carry our three plasmid co-transformation</p>
 +
<p>Add tag to GFP</p>
 +
 +
 +
 +
<p><b>SUM UP</b></p>
 +
<p>Confirm the three antibiotic concentration and get photo of the experiment result. </p>
 +
<p>Perform co-transformation of the B2H strains. </p>
 +
<p>Meet some problem when culturing the strains in three antibiotic culture. </p>
 +
<p>Finishing preparing the B2H strains. </p>
 +
<p>Prepare positive control for fluorescent test (strain that can express RFP and YFP respectively) </p>
 +
<p>Due to the plasmid of plac is somehow degredation, plac related experiment failed</p>
 +
<p>pSB1C3-Or2-GFP is successfully constructed</p>
 +
<p>Finish preparing competent cells</p>
 +
<p>Get plac-YFP as positive control. </p>
 +
<p>Get GFP co-transformation strains. </p>
 +
<p>Decided it is not necessary to add tag to GFP. </p>
 +
 +
 +
<h2><b>AUGUST</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>Design primers for mutants</p>
 +
<p>Sequencing for pSB4A5-Or2</p>
 +
<p>Check the C+ plate. </p>
 +
<p>Test the inducement completely. </p>
 +
<p>Go to TW for the meet up</p>
 +
<p>Li Pai continue to repeat the B2H experiments</p>
 +
<p>Prepare new BL21 competent cells ( the strain from 4th floor with lower leaky expression) </p>
 +
<p>Confirm the colony of BL21 can be induced. </p>
 +
<p>Continue to repeat the induction experiments</p>
 +
<p>Decide whether it is necessary to use the reporter strain</p>
 +
<p>Using BL21 from 4th floor to obtain co-transformation strains</p>
 +
<p>See whether reporter cells can be infected by M13 phage? (how?) </p>
 +
 +
 +
 +
<p><b>SUM UP</b></p>
 +
<p>Mutation of LGF2 and Inducement. </p>
 +
<p>Solve the problem that the measuring of RFP expression strain using OD 600 is not that exactly. </p>
 +
<p>Confirmed that the colony of BL21 can be induced. </p>
 +
<p>Repeated the induction experiments</p>
 +
<p>BL21 we prepared ourselves have leaky expression, but the origin doesn't</p>
 +
 +
 +
<h2><b>SEPTEMBER</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>Use the confirmed strains to test fluorescents
 +
Draw the graph
 +
Plan for flow cytometry ( buy beats?) </p>
 +
<p>Plan for part submission
 +
Design primers for λcI-LGF2 mutants
 +
Plasmid extraction of Or2? </p>
 +
<p>Integrate the system.
 +
Design the strategy to integrate B2H system to other parts. </p>
 +
<p>Finish Biobrick vector construction</p>
 +
<p>Finish point mutation experiment and test fluorescent</p>
 +
<p>Start doing western blot</p>
 +
<p>B2H system integrated with RNAT</p>
 +
<p>Test fluorescent of HML groups</p>
 +
 +
 +
 +
<p><b>SUM UP</b></p>
 +
<p>Tested  the fluorescents and draw the graph. </p>
 +
<p>Integrated the system. </p>
 +
<p>Constructed the Biobrick vector. </p>
 +
<p>Finish point mutation experiment and test fluorescent</p>
 +
<p>Western blot</p>
 +
<p>B2H system integrated with RNAT</p>
 +
<p>Fluorescent of HML groups tested. </p>
 +
 +
 +
 +
<h2><b>OCTUBER</b></h2>
 +
<p><b>PLAN</b></p>
 +
<p>Quick change for the λcI-LFG2</p>
 +
<p>Test fluorescent of Or2-RNAT groups</p>
 +
<p>Prepare for western of pSB4A5-Or2-p8</p>
 +
<p>Try p3 again</p>
 +
<p>Prepare for Gal11P-RNAα integrated into M13 plasmid</p>
 +
<p>IPTG induction</p>
 +
<p>P3 related strains spread the plate</p>
 +
<p>Prepare to amplify the pBT-LGF2 plasmid</p>
 +
 +
 +
 +
<p><b>SUM UP</b></p>
 +
<p>The 1000 group is significantly lower than 10 group</p>
 +
<p>Test fluorescent of Or2-RNAT groups</p>

Revision as of 20:41, 17 October 2014

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.


OCTUBER

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

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