Team:HKUST-Hong Kong/notebook.html

Notebook

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 (13rd-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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