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Notebook

• Genomic DNA of E.coli DH10b extracted
• Culture Tests:
• Waiting for materials to arrive

Week 2 (13th-17th June)
Strain construction:

• Genomic DNA of E.coli  DH10b extracted Genomic DNA of BL21 extracted
• Secured split superfolder GFP construct transformed out
• Finished design of PCR primers
• Culture Tests:
• Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)
• Constructed standard curve for OD600 verses RR1 CFU concentration

Week 3 (20th-24th June)
Strain construction:

• nadE: PCR out nadE gene from the genome of BL21
• Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.
• 2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into E.coli DH10b. Test will be conducted soon.
• Lambda-RED and oriR101&repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready.

Culture Tests:

• Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals)
• Miniprep of BBa_I763007 and BBa_E1010 was successful

Week 4 (27th June – 1st July)
Strain construction:

• Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful
• 2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP
• Split superfolderGFP system: protocol design finished, primer arrived
• Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful

Culture Tests:

• Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007)
• Transformation of the RFP/KanR plasmid to E.coli DH10b

Week 5 (8th-12th July)
Strain construction:

• ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration
• pir gene: gDNA of BW25141 extracted
• Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed
• 2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein
• oriR101&repA101-ts: PCR was successful

Culture Tests:

• Successful construction of a RFP-labeled kanamycin-resistant strain .
• Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function.
• MIC testing for RR-1

Week 6 (15th-19th July)
Strain construction:

• Lambda RED: RFP with homologous sequence PCR successful
• 2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished
• Split superfolderGFP system : PCR of spilt superfolderGFP successful
• 2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo
• pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-gamma, the sequence PCR of the pir gene is done, wait to check the result
• nadE gene: ligate nadE gen with double terminator, not successful, do another try

Culture Tests:

• MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals)
• MIC test for mixed cultures of RFP/KanR and RR1(1:99)
• Literature search – multidrug pump candidates
• Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold
• NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell

Week 7 (22th-26th July)
Strain construction:

• pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected
• Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification
• 2010 Slovenia’s method- CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed à no fluorescence
• nadE gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick
• oriR101&repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR
• Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress
• pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit

Culture Tests:

• Mixed culture MIC tests for RFP/KanR and RR1(1:99)
• Multidrug Efflux Pump – Settled on Bcr as the candidate gene
• 2~4 folded increasing for Kan
• Proton gradient (H+) driven
• Pumps out other toxins
• Unknown promoter
• E.coli DH10a containing pUC18not/T4MO arrived.

Week 8 (1st-5th Aug)
Strain construction:

• pir gene: Sequencing result has just come out
• Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing
• 2010 Slovenia’s method- CFP/YFP: Finished construction but not verified
• nadE gene: finished
• oriR101&repA101-ts: Been ligated to a backbone, verifying
• Lambda RED: Waiting for the primers to construct the linear sequence

Culture Tests:

• Completed the standard curve for OD 600 versus RFP/KanR CFU concentration
• Mixed culture MIC tests for RFP/KanR and RR1(1:99)
• Successfully extracted T4MO from pUC18not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly.
• PCR amplified bcr gene from gDNA of E. coli stock

Week 9 (8st-12th Aug)
Strain construction:

• pir gene: Exact location of pir gene in BW25141 is mapped out
• Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week
• 2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing
• oriR101&repA101-ts: Verifying oriR101&repA101-ts
• Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene
• pCarrier: MCS is hybridized. pSB1K3 is under digestion

Culture Tests:

• Digestion of T4MO/pBS KS+ failed
• Successfully ligated bcr gene with RBS (later confirmed to be false positive)

Week 10 (15st-19th Aug)
Strain construction

• pir gene: Ligation done and being verified
• Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.
• Lambda RED: The swapping seemed to be successful
• oriR101&repA101-ts: Waiting for new primers
• pCarrier: MCS and OriR ligated

Culture Tests:

• Indole MIC test for wild type (1mM with kanamycin gradient):
• Successfully ligated T4MO into pBS KS+

Week 11 (22st-26th Aug)
Strain construction:

• pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone
• Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation
• 2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.
• nadE gene: Complete.
• oriR101&repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.
• pToolkit construction: Complete
• pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress

Culture Tests:

• Indole MIC test (500µM with kanamycin gradient)
• Ligation of the RBS+Bcr with pLac promoter failed

Week 12 (29st Aug- 1th Sep)
Strain Construction

• pir gene: Background self-ligation is under test, results will be available tomorrow
• Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with lacpromoter
• 2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done;
• nadE gene: Completed; veri;
• oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3;
• lambda RED:check whether swap is successful: screen 6 colonies for verification;
• pToolkit construction: Complete
• pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS

Culture Tests

• Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
• Indole MIC test (300µM with kanamycin gradient)
• Successfully ligated T4MO with GFP

Week 13 (5th-9th Sep)
Strain construction

• Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived
• oriR101&repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr
• Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick.
• pir gene and ori-gamma: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets).
• nadE gene: Completed; wait to do sequencing verification;
• pCarrier: MCS reinsert, change the size and position of insertion;
• pToolkit construction: accidentally disappear, redo the whole plasmid;
• pCarrier: nadE part ready, working on MCS now.

Culutre Test

• Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
• Indole MIC test (1mM indole concentration with kanamycin gradient)
• Successfully ligated T4MO/GFP into kanamycin resistant backbone.

Week 14 (13th-17th Sep)
Strain Construction:

• lambda RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E.COLI DH10B still have some bands); consider directly PCR out from pKD46
• oriR101&repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress
• Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick;
• pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing;
• nadE gene: Completed; wait to do sequencing verification
• pToolkit construction: accidentally lost, redo the whole thing
• pCarrier : MCS insertion does not show good result halted for this year

Culture Test

• Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)
• Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed]
• Started to construct Biobrick of bcr gene for submission

Week 15  (20th-24th Sep)
Strain Construction;

• oriR101&repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week
• pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation) or star activity; insert the pir to pBS again (use different enzymes), sequence again.
• Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress

Week 16 ( 27th- 30th Sep)
Strain Construction

• oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed;
• Split superfolderGFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence
• pir gene: sequence failed (wrong gene…nadE actually)