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Week 1 (4th-10th June)
Strain construction
- Genomic DNA of E. coli DH10b extracted
- Waiting for materials (bacterial strains, primers) to arrive
Week 2 (13th-17th June)
Strain construction
- Genomic DNA of E. coli DH10b extracted. Genomic DNA of BL21 extracted
- Cloned out split superfolder GFP construct
- Finished design of PCR primers for nadE gene and λ RED
Culture Test
- Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)
- Constructed standard curve for OD600 verses RR1 (wild type) CFU concentration
Week 3 (20th-24th June)
Strain construction
- nadE: PCR out nadE gene from the genome of BL21
- Split superfolder GFP system: The test of the intact (sfGFP 1-10 still ligated together with sfGFP11) superfolder GFP was successful. Confirmation tests and further experiments would be conducted soon.
- 2010 Slovenia’s method - CFP/YFP: BioBricks would be transformed into E. coli DH10b. Tests would be conducted soon.
- λ RED and oriR101&repA101-ts: pKD46 has arrived and successfully extracted. RFP reporter system was ready. Primers of RepA101ts-OriR101 are ready.
Culture Test
- Performed 2nd and 3rd MIC tests for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals)
- Minipreps of BBa_I763007 and BBa_E1010 were successful
Week 4 (27th June–1st July)
Strain construction
- λ RED : protocol design finished; pKD46 arrived and digestion tests indicated that the plasmid was correct; PCR RFP with homologous sequence was successful
- 2010 Slovenia’s method - CFP/YFP: protocol design finished, successfully finished combined CFP and YFP
- Split superfolder GFP system: protocol design finished; primer arrived
- Pir gene and ori-γ: protocol under construction; primer for ori-γ arrived; BW25141 gDNA extraction successful
Culture Test
- Ligation of pSB2K3 (from BBa_E1010) with RFP reporter device (BBa_I763007)
- Transformation of the RFP/KanR plasmid to E. coli DH10b
Week 5 (8th-12th July)
Strain construction
- ori-γ: Primers arrived, PCR was successful. Result: one of four samples was positive, but of low concentration
- pir gene: gDNA of BW25141 successfully extracted
- Split superfolder GFP system: GFP1-10 PCRed, GFP11 PCRed
- 2010 Slovenia’s method - CFP/YFP: Verified combined fluorescence protein
- oriR101 & repA101-ts: Clone- out by PCR was successful
Culture Test
- Successful construction of a RFP-labeled kanamycin-resistant strain
- Information from literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly help it survive in kanamycin long enough to fulfill its function
- MIC testing for RR-1
Week 6 (15th-19th July)
Strain construction
- λ RED: PCR of RFP with homologous sequence successful
- 2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript KS+ promoter finished
- Split superfolder GFP system: PCR of spilt superfolder GFP successful
- 2010 Slovenia’s method - CFP/YFP :Ligation with promoter is successful, but the green fluorescence could not be observed. Considering to redo construction
- pToolkit construction: PCR of ori-γ successful; ligation with pKD46 backbone was done, confirmation still awaiting the results of colony PCR to check the existence of ori-γ; the sequencing PCR of the pir gene was done, now waiting to check the results
- nadE gene: ligation nadE gene with double terminator not successful. Would repeat experiments next week
Culture Test
- 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): overexpression reduces radical oxidative species (e.g. H2O2) inside the cell
Week 7 (22nd-26th July)
Strain construction
- pir gene: Sequencing product did not meet sequencing requirement (a lot of 'N's) – sequencing rejected. To do: practice how to perform sequencing clean-up properly
- Split superfolder GFP system: Construct to be ligated had very low recovery from gel purification; another trial would be done asap
- 2010 Slovenia’s method – CFP/YFP: combination of n-terminal and c-terminal CDS into same plasmid, driven by plac promoter of pBluescriptKS+ completed. Result: very weak fluorescence observed
- nadE gene: successful ligation of nadE gene with terminator; correctness of construct confirmed by restriction digestion tests. Component was putatively finished as biobrick
- oriR101&repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR
- λ RED: Previous experiment of gene swapping failed. Trouble-shooting in progress
- pToolkit construction: results from colony PCR of ori-γ from transformed bacteria: successful completion of pToolkit. Further confirmation by restriction digestion to be done
Culture Test
- Mixed culture MIC tests for RFP/KanR and RR1(99:1)
- 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 superfolder GFP 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: construction finished, but construct was still harbored in pSB1AK3. Consider relocation to pSB1C3 asap.
- oriR101&repA101-ts: Been ligated to a backbone, verifying
- λ RED: Waiting for the primers to construct the linear dsDNA sequence (for swapping)
- pCarrier: Design of Multiple Cloning Site sequence had been completed, waiting for oligos to arrive.
Culture Test
- Completed the standard curve for OD600 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 (8th-12th Aug)
Strain construction
- pir gene: Exact location of pir gene in BW25141 is mapped out
- Split superfolder GFP system: Primers did not work: a lot of non- specific bindings, expected band size was not clearly present. New primers had been designed; waiting for new primers to come next week
- 2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP still on progress
- oriR101&repA101-ts: Verifying oriR101&repA101-ts
- λ RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene
- pCarrier: MCS had been hybridized. pSB1K3 is under digestion
Culture Test
- Digestion of T4MO/pBS KS+ failed
- Successfully ligated bcr gene with RBS (later confirmed to be false positive)
Week 10 (15th-19th Aug)
Strain construction
- pir gene: Ligation done and being verified
- Split superfolder GFP system: PCR with new primers. Split superfolder GFP11 digested and ligated with the promoter.
- λ RED: The swapping seemed to be successful
- oriR101&repA101-ts: Waiting for new primers
- pCarrier: MCS and pSB1AK3 with nadE ligated, but not confirmed
Culture Test
- Indole MIC test for wild type (1mM with kanamycin gradient):
- Successfully ligated T4MO into pBS KS+
Week 11 (22nd-26th Aug)
Strain construction
- pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone
- Split superfolder GFP 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 λ 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 Test
- Indole MIC test (500µM with kanamycin gradient)
- Ligation of the RBS+Bcr with pLac promoter failed
Week 12 (29th Aug-1st Sep)
Strain Construction
- pir gene: Background self-ligation is under test, results will be available tomorrow
- Split superfolder GFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with lac promoter
- 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; verified;
- oriR101 + repA101ts: Construction is complete – verified by restriction digestion; BioBrick currently located on pSB1AK3;
- λ 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 Test
- 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
- λ 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 superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick.
- pir gene and ori-γ: 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
- λ 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 superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick;
- pir gene and ori-γ: 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 key parts are missing. the target part developed an unexpected illegal cut (point mutation or star activity); insert the pir to pBS again (using different enzymes), sequenced again.
- Split superfolder GFP 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 superfolder GFP 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)
- pToolkit construction: construction in progress
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