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

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• 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.

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 pSB1A2 (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
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agtc

This information is also recorded on page 11 of the hard copy lab notebook.
• Wednesday 6/16--checked on spotted cell survival assay, collected MOPS minimal media materials, started making component solutions
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• Thursday 6/17--more minimal media work and meeting, started BL21 culture
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• Friday 6/18--plasmid pSB74 arrived (!), already in some unspecified E coli, so plated them out. Transformed promoters (constitutive & IPTG inducible), terminator, and repressor (for inducible promoter) into BL21. Also ran BL21 copper growth assays for wide and narrow concentrations ranges. June the 19th--in which it is revealed that there was a transformation-fail Redid transformation, this time into LE392, set aside pSB74 containing cells in fridge
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• Sunday 6/20--in evening inoculate 5 mL liquid cultures
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