Team:Yale/Our Project/Notebook/Week 2

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lab notebook: week 2 (6/14-6/20)

• Monday 6/14--Redo of 6/11 assay of bacterial growth within a narrow copper(II) concentrations after analysis of data showed uniformly poor growth.
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Copper Growth Assay Work Continues



• As the growth assay of 6/11 showed uniformly poor growth even at the lowest copper concentrations (see Dh5alpha growth above), redid it with care not to let the liquid cultures overgrow, a possible source of the cultures' previous poor performance. (add plot)
• In redoing plate assay, changed copper concentration line-up slightly, eliminating 1.25 mM and 2.5 mM trials to allow room for the control concentrations, 0 and 1 M, and only worked with high because there was no real distinction in the three different OD trials done on 6/10. For simplicity, future assays will only use one starting OD (0.075) per strain.
• During the repeat, noticed that DH5alpha liquid culture grown up for plate reader assay grew at a rate much slower than that of LE392, creating a need to keep diluting the LE392 solution with more LB so it would not overgrow before the DH5alpha was ready. In the end, the culture of LE392 used in the assay was diluted from an OD of 0.761 and the DH5alpha culture from an OD of 0.841.

This day's work is also recorded on pages 8-10 of the lab notebook hard copy.
• Tuesday 6/15--Results and analysis of the 6/14 narrow concentration range growth assay as well as planning for the creation of a standard iGEM plasmid bearing the thiosulfate reductase operon (phsABC).
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Results and Conclusions of Narrow Concentration Range Growth Assay of 6/14



• Negative readings for the 4 mM samples suggest that there was some sort of irregularity with the blank solution, so will disregard those results.
• Can see growth really starts to slow down at 3 mM concentrations of Cu²⁺
• It’s unclear whether the cells at the higher copper concentrations die or simply stop growing. Spot a drop from each well of the plate onto a LB agar plate and incubate overnight at 37˚C. Will check for growth to determine survival of different strains at different copper concentrations.

This information is also recorded on page 11 of the hard copy lab notebook.


Plasmid Ligation Strategy

The Plan for phs plasmid creation
The phs operon has three genes of interest to us, A, B, and C, that code for different protein products and together are responsible for hydrogen sulfide production. We will use these genes to create a number of different plasmids described below.
Plasmids to be made:
Plasmid 1 gives our basic thiosulfate reductase pathway under IPTG control. Plasmid 5 is a light-controlled analog that we will make if we can find a suitable light-inducible promoter. Plasmid 2 is a promoter-less “generator” to be archived so other teams can use it. Plasmids 3 & 4 are a set and together give the entire pathway. The idea is that since protein production takes a while we will make only part of the pathway inducible. The cell will constitutively produce the A & B products, so after the light hits only the small C protein remains to be made.


Product	Promoter	Gene	Terminator	Plasmid Vector
1	IPTG-inducible BBa_R0011(well 6G plate 1)	phsABC	BBa_B0015	pSB1C3 (chloramphenicol resistance)
2	none)	phsABC	BBa_B0015	pSB1C3 (chloramphenicol resistance)
3	Constitutive BBa_J23114 (well 20I plate 1)	phsAB	BBa_B0015	pSB1T3 (Tet resistance)
4	Light-inducible	phsC	BBa_B0015	pSB1C3 (chloramphenicol resistance)
1	light-inducible	phsABC	BBa_B0015	pSB1C3 (chloramphenicol resistance)

*This plan was eventually altered, as promoter choices were altered and no light-inducible promoter was found.





To make these plasmids, we will rely on the standard iGEM assembly protocol involving restriction enzymes EcoRI, XbaI, PstI, and SpeI shown in the diagram at right, but in the first ligation the B0015 terminator will take the place of C0010 and the phsABC gene in pSB74 will take the place of B0034.
Steps to make these plasmids
1. Initial Amplification—transform and amplify terminator, promoter, and gene DNA
2. Gene-Terminator linkage—Digest terminator with EcoRI and XbaI, digest PCR product gene with EcoRI and SpeI, and ligate together to get gene and terminator in nonstandard plasmid
3. Creation of Standard Generator—digest step 2 product with EcoRI and PstI and ligate into standard iGEM plasmid pSB1C3 to get the generator
4. Addition of promoter—Digest promoter with EcoRI and Spe--digest generator vector with EcoRI and XbaI, and ligate together


Primer design for PCR amplification of the phsABC gene from background vector pSB74

Given the size of phsABC, introducing it in pieces with AB on one plasmid and C on a second might enhance expression. For this purpose, we have designed forward primers that include the EcoRI and XbaI cut sites and an RBS to go at the beginning of A and C as well as reverse primers with a SpeI site to go at the end of B and C.
The following primers were designed based on the phsABC sequence information (see below) after having confirmed that the BioBrick restriction enzyme cut sites are not present in the phsABC sequence.
N-terminal primer for phsABC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AGC ATT AGT CGT CG
C-terminal primer for phsAB: 5’ AGC ACTAGT A TCA TGC TTT ACC CTC CTG ATG GAC
N-terminal primer for phsC: 5’ GCA GAATTC GCGGCCGC T TCTAGA G AGGAGGTTTAT ATG AAT ACT ATC TGG GG
C-terminal primer for phsABC: 5’ AGC ACTAGT A TCA GAC GGC GGA CTT ATC CCC
See/hide phsABC sequence

phsA STM2065
>sp|P37600|PHSA_SALTY Thiosulfate reductase OS=Salmonella typhimurium GN=phsA PE=3 SV=2
MSISRRSFLQGVGIGCSACALGAFPPGALARNPIAGINGKTTLTPSLCEMCSFRCPIQAQ VVNNKTVFIQGNPSAPQQGTRICARGGSGVSLVNDPQRIVKPMKRTGPRGDGEWQVISWQ QAYQEIAAKMNAIKAQHGPESVAFSSKSGSLSSHLFHLATAFGSPNTFTHASTCPAGKAI AAKVMMGGDLAMDIANTRYLVSFGHNLYEGIEVADTHELMTAQEKGAKMVSFDPRLSIFS SKADEWHAIRPGGDLAVLLAMCHVMIDEQLYDASFVERYTSGFEQLAQAVKETTPEWAAA QADVPADVIVRVTRELAACAPHAIVSPGHRATFSQEEIDMRRMIFTLNVLLGNIEREGGL YQKKNASVYNKLAGEKVAPTLAKLNIKNMPKPTAQRIDLVAPQFKYIAAGGGVVQSIIDS ALTQKPYPIKAWIMSRHNPFQTVTCRSDLVKTVEQLDLVVSCDVYLSESAAYADYLLPEC TYLERDEEVSDMSGLHPAYALRQQVVEPIGEARPSWQIWKELGEQLGLGQYYPWQDMQTR QLYQLNGDHALAKELRQKGYLEWGVPLLLREPESVRQFTARYPGAIATDSDNTYGEQLRF KSPSGKIELYSATLEELLPGYGVPRVRDFALKKENELYFIQGKVAVHTNGATQYVPLLSE LMWDNAVWVHPQTAQEKGIKTGDEIWLENATGKEKGKALVTPGIRPDTLFVYMGFGAKAG AKTAATTHGIHCGNLLPHVTSPVSGTVVHTAGVTLSRA

>phsA
atgagcattagtcgtcgttcttttttgcagggggtaggcatcggctgctctgcctgcgcg ctgggcgcttttccgcccggggcgctggcgcgcaacccgattgccggcattaacggtaaa accacgctaacgccaagcttgtgcgaaatgtgttcctttcgttgccctattcaggcgcag gtagtcaataacaagaccgtttttatccagggcaatccttctgcgccgcagcaggggacg cgcatatgcgccaggggcgggagcggcgtcagcctggtcaatgacccgcaacggattgta aaacctatgaaacgcaccgggccacgcggcgacggtgaatggcaggtgattagctggcaa caggcttaccaggaaatcgcggcgaaaatgaatgccatcaaagcgcagcatggccccgag agcgtggctttctcttccaaatcgggctcgctctccagccatcttttccatctggctacg gcctttggttcgcctaatacctttacgcacgcctcaacatgccctgccgggaaagccatt gcggcaaaagtgatgatgggcggcgatctggcgatggatatcgctaacacgcgctatctg gtttcgtttggccacaatttgtatgaagggattgaagttgccgatacccatgagttaatg accgcgcaggagaagggggccaaaatggtgagcttcgatccgcgtttgtcgatattttcc agcaaggcggatgagtggcacgctattcgtcccgggggggatttagcggttctgctggcg atgtgccacgtcatgattgatgaacagctctacgatgcgtcttttgttgagcgttatacc agcggatttgaacagttagcacaggcggtaaaagagacgacgccggaatgggccgccgcg caggccgatgtcccagccgacgttattgtccgggtgacacgcgaactggctgcctgcgcg cctcacgctattgtcagtcctggtcatcgcgcgacgttctcgcaggaagagatcgatatg cggcgtatgatttttacgcttaatgtgctgctcggtaatattgagcgcgaaggcgggcta tatcagaaaaaaaacgcgtctgtttacaataaactggccggagaaaaggtcgcgccaacg ctggcgaaactcaacattaaaaatatgccgaaaccgacggcgcaacgcatcgatttggtc gcaccgcagtttaaatatatcgccgctggcggcggcgtggtgcaaagcattattgactcg gcgttaacccagaagccttacccgataaaggcgtggattatgtcgcggcataatcctttc cagaccgtcacctgtcgttcggacctggtaaaaaccgttgagcaactggatctggtggtc agctgcgatgtctatttgagcgagagcgcggcatatgccgactatctgctgccggaatgc acctatctcgaacgggacgaagaggtatccgatatgtcgggactgcacccggcttacgct ctgcgccaacaggtcgtagagccgattggcgaggcgcgtccgagttggcaaatctggaaa gaacttggcgagcagttgggattagggcagtactatccgtggcaggatatgcagacgcgc caactctatcagttgaacggcgaccatgccttagcgaaggaactgcgacagaaagggtat ctcgaatggggcgttccgctgctattacgcgaaccagaatccgttcgtcagtttacggcg cgttaccccggcgctatcgcgacggacagtgacaacacctatggcgaacagcttcgcttc aaatcgccctccggcaaaatcgaactttattccgcaaccctggaggaattgctccctggc tacggcgttccgcgcgttcgtgactttgcgctgaaaaaagagaatgagctttacttcatt cagggcaaggtggccgtgcataccaatggcgcgacgcagtacgtacctttactcagcgag ctaatgtgggataacgcggtctgggttcatccgcaaacggcgcaagaaaaaggcattaag accggcgatgagatctggctggaaaatgccacgggtaaagagaaaggtaaggcgctggtg acgcccggtatccgcccggacacgctttttgtctatatgggatttggcgctaaagctggg gccaaaacggcggcgacgacacacggtatccactgcggaaatttactgccgcacgtgacg agtccggtatccggtacggtagtgcataccgcaggcgtgacgctgagccgggcatga

phsB STM2064
>sp|P0A1I1|PHSB_SALTY Thiosulfate reductase electron transport protein phsB OS=Salmonella typhimurium GN=phsB PE=4 SV=1
MNHLTNQYVMLHDEKRCIGCQACTVACKVLNDVPEGFSRVQVQIRAPEQASNALTHFQFV RVSCQHCENAPCVSVCPTGASYRDENGIVQVDKSRCIGCDYCVAACPFHVRYLNPQTGVA DKCNFCADTRLAAGQSPACVSVCPTDALKFGRLDESEIQRWVGQKEVYRQQEARSGAVSL YRRKEVHQEGKA

>phsB
Atgaatcatttaacgaatcagtacgtcatgctgcatgatgaaaaacgttgtatcggctgc caggcgtgtaccgttgcctgcaaagtgcttaatgacgtaccggagggatttagccgcgta caggtacaaattcgcgcccctgaacaggcatccaacgcactaacccattttcaatttgtc cgcgtctcctgtcagcactgtgaaaatgcgccatgtgtcagcgtttgtcctaccggagcg tcatatcgtgatgaaaacgggatcgtgcaggtggataaatcgcgctgtattggctgcgat tattgtgttgccgcgtgtcctttccatgtgcgctatttgaatccgcaaaccggtgtcgcc gacaagtgtaacttctgcgccgacacgcggttggcggctggccagtctccggcgtgcgta tccgtttgcccaacggacgcgctgaaattcggcagactggatgagagcgagatccagcgc tgggtcggtcagaaagaggtctatcgccagcaggaggcgcgtagcggcgcggtcagtctg taccgtcgtaaagaagtccatcaggagggtaaagcatga

phsC STM2063
>sp|P37602|PHSC_SALTY Thiosulfate reductase cytochrome B subunit OS=Salmonella typhimurium GN=phsC PE=4 SV=2
MNTIWGAELHYAPDYWPLWLIYAGVVVLLMLVGLVIHALLRRMLAPKTAGGEEHRDYLYS LAIRRWHWGNALLFVLLLLSGLFGHFSLGPVALMVQVHTWCGFALLAFWVGFVLINLTTG NGRHYRVNFSGLVTRCIRQTRFYLFGIMKGEAHPFVATEQNKFNPLQQLAYLAIMYALVP LLIITGLLCLYPQVAGLGPVMLVLHMALAIIGLLFICAHLYLCTLGDTPGQIFRSMVDGY HRHRTAPRGDKSAV

>phsC
Atgaatactatctggggagcggaactacattatgcgccagattattggccgctgtggttaa tttacgcaggcgtcgtggtgctgctcatgcttgttgggctggttatccatgcgttattgcg ccggatgctggcgccaaaaacggcgggcggtgaagaacatcgtgactatctctactcgctg gcgattcgccgctggcattggggaaatgcgttactgtttgttttattactgttaagcggtt tatttggtcatttttctctcggccctgtagcgctaatggtacaagtgcatacctggtgtgg ttttgccttactggctttctgggtcgggtttgtgctgatcaacctcaccacaggtaacggg cgtcactatcgggtaaatttttccggactggtaacgcgctgcatacgccagacgcgttttt acctttttggcattatgaaaggggaagcgcatccgttcgtggcaacagagcagaataagtt caatccactgcaacaactggcatatctggcgattatgtacgcgctggtaccgctgttaatc atcaccggtttgctgtgtctctatccgcaggttgcgggtctgggccctgtgatgctggtgc tgcatatggcgcttgctatcatcggcttactgtttatttgcgcgcatctctatctgtgtac tcttggcgacacgccgggacaaattttccgtagcatggttgacggctatcatcgtcatcgt accgcgccgcgcggggataagtccgccgtctga




• Wednesday 6/16--Checked on spotted cell survival assay, collected MOPS minimal media materials & started making component solutions
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Minimal Medium Work
In Jay Keasling's work with thiosulfate reductase he used a variation of MOPS minimal medium designed to avoid background metal presence from interfering with measurement of bacterial metal removal potential. Began making the various necessary component solutions as detailed in this recipe
Survival of bacteria in copper growth assay
Previously had plated a drop of each culture from the narrow concentration range copper growth assay on 6/14 to see if the cultures that had stopped growing/never grew were still alive. Observed the following results, where g signifies growth and - signifies no growth:

cell line

0 M Cu2+

500 uM Cu2+

600 uM Cu2+

700 uM Cu2+

800 uM Cu2+

900 uM Cu2+

1 mM Cu2+

1.5 mM Cu2+

2 mM Cu2+

3 mM Cu2+

4 mM Cu2+

1 M Cu2+

DH5alpha

-

g

g

g

g

-

g

-

-

g

g

-

LE392

g

g

g

g

g

g

g

g

g

g

g

-
It is unsurprising that all cells should die at 1 M copper sulfate concentrations, but the lack of growth of DH5alpha at 0 M, 900 uM, etc. seems anomalous, given that the cells clearly survived at higher copper levels and may reflect an insufficient amount of culture spotted in those cases.
This information is also recorded on page 11 of the hard copy lab notebook.

• Thursday 6/17--more minimal media work and meeting, started BL21 culture
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Modified MOPS minimal medium
Finished creation of a stock solution according the this recipe
Additional E. coli strain
Began a culture of BL21 as another hardy option for future assays.

• Friday 6/18--Arrival of plasmid pSB74, transformation of Biobricks, & copper growth assays for BL21 strain.
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• Plasmid pSB74, which contains the thiosulfate reductase gene, arrived from Addgene, already in some unspecified E coli, so plated them out on an ampicillin LB plate and put in incubator at 37.
• Transformed constitutive promote J23114, IPTG-inducible promoter R0011, terminator B0015, and repressor C0012(for inducible promoter) into BL21 according to standard transformation protocol and plated onto ampicillin LB plates
• Also ran BL21 copper growth assays for wide and narrow concentrations ranges according to the modified protocol used on 6/14, starting with dilution of a culture of OD 0.873.
The activities of this day are also recorded on pages 13-15 of the hard copy lab notebook
• Saturday 6/19--Redo of Biobrick transformations & analysis of BL21 copper growth assay
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Transformation troubles
While the pSB74 culture grew, none of the BL21 transformants from 6/19 did, so repeated the transformations of Biobricks B0015, R0011, C0012,and J23114 into LE392 cells, once again following this transformation protocol and plating onto ampicillin LB plates. The remaining transformant solutions were set aside in case of another failure.
BL21 copper growth assay analysis
Retrieved the following data regarding BL21's growth in copper solution:


It appears that BL21 is slightly more sensitive to copper than the other two strains, since 3 mM levels of copper(II) sulfate are enough to almost completely inhibit BL21's growth.
The activities of this day are also recorded on page 15 of the hard copy lab notebook
• Sunday 6/20--Observed growth of all transformants from 6/19, so used them and the culture containing pSB74 to inoculate 5 mL liquid cultures in LB with ampicillin. Left to grow overnight on shaker at 37˚C for miniprep the following morning.
  The activities of this day are also recorded on page 16 of the hard copy lab notebook