Team:William and Mary/Notebook


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Notebook

Important Protocols

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UNS Gibson

for building constructs

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Iterative Capped Assembly

for assembling repeated monomeric sequences of arbitrary length

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small molecule induction

for inducing protein expression

flow cytometry

for measuring fluoresence at single-cell resolution

Week by Week Summary

Week 1 (160529 – 160604)

Resuspended parts from the kit.

Resuspended gBlocks of ordered sequences.

Created a functional UNS Standard Backbone, containing the UNS 2 and 3 sequences within the Prefix and Suffix.

Cloned K2066001-K2066016 into UNS pSB1X3 Backbone.

Performed Diagnostic PCRs to test primer design for Ribozyme / RiboJ Characterization subproject

Transformed interlab devices, created glycerol stocks. Could not get IMP #1, #3, and (-) control to transform.

Week 2 (160605 – 160611)

Constructed K2066024, K2066025, and K2066027 using Gibson Assembly (from K2066014, K2066015, and pTAC templates).

We received training on the FACS (Fluorescence Activated Cell Sorter) Machine.

Realized the need for repressor protein / fluorophore fusion proteins (LacI-mCherry, TetR-GFP, i.e.)

Designed gBlocks of K2066028 and K2066029.

Attempted to clone Addgene 240x TetO Sequence into UNS Standard Backbone.

Attempted ICA with K2066002, K2066003, K2066004 to create a TetO w/ 8bp spacer 9-mer

Constructed K2066030 and K2066031 from K2066015.

Troubleshot ICA, attempted 6- and 12-mers of TetO w/ 8bp spacer.

Attempted IPTG Induction of K2066014 + K2066016 cotransformations.

Transformed remaining interlab devices, created glycerol stocks.

Week 3 (160612 – 160618)

Assembled K2066026, K2066030, K2066031 using Gibson assembly.

Verified fidelity of cotransformations using colony PCR.

Continued to troubleshoot ICA: attempted 2-mer, failed.

Tested Arabinose Induction on K5577882, pBAD-RFP.

Verified ability of BsmBI to cut ICA Monomers.

Troubleshot induction: tried various OD-600s, strains, and plasmid copy numbers.

Week 4 (160619 – 160625)

Moved K2066014, K2066015, K2066027, K2066031, K2066016 onto various low copy number backbones to test effect of copy number on induction.

Performed cotransformations of K2066014, K2066016 with many different copy number variations.

Switched to Benchling.

Week 5 (160626 – 160702)

Assembled 85x tetO Array on 1C3.

Moved K2066024 and K2066026 to low copy backbones.

aTc and Arabinose inductions of pTet GFP and pBad RFP constructs, flow cytometry analysis.

Created electrocompetent lg3.300 cells (strain used for synthetic enhancer characterization).

Received synthetic enhancer parts from Roee Amit’s lab.

Decided to cut ICA from project.

Week 6 (160703 – 160709)

Cotransformations of pTet GFP and TetR constructs.

Tested fidelity of various restriction enzymes’ ability to cut 240x and 85x TetO arrays.

Cotransformations of 57S + pACT-Tet into LG3.300 to test induction in context of synthetic enhancer constructs.

Attempted to make electrocompetent BL21 cells, unfortunately results were low quality and fairly unable to transformation.

Attempted assemblies to move synthetic enhancer parts onto BioBrick backbone

Restriction digest successfully moved 85x TetO Array, but it was moved into the Prefix of K2066014 instead of the standard position of between the Prefix and Suffix. Will have to try a different approach.

Assembled and transformed permutations of synthetic enhancer parts on low copy number plasmids to test effect on induction.

Remade 85x TetO Array on BioBrick backbone using different restriction enzymes.

● Received bacterial Broccoli from Addgene.

Week 6 (160703 – 160709)

Cotransformations of pTet GFP and TetR constructs.

Tested fidelity of various restriction enzymes’ ability to cut 240x and 85x TetO arrays.

Cotransformations of 57S + pACT-Tet into LG3.300 to test induction in context of synthetic enhancer constructs.

Attempted to make electrocompetent BL21 cells, unfortunately results were low quality and fairly unable to transformation.

Attempted assemblies to move synthetic enhancer parts onto BioBrick backbone

Restriction digest successfully moved 85x TetO Array, but it was moved into the Prefix of K2066014 instead of the standard position of between the Prefix and Suffix. Will have to try a different approach.

Assembled and transformed permutations of synthetic enhancer parts on low copy number plasmids to test effect on induction.

Remade 85x TetO Array on BioBrick backbone using different restriction enzymes.

Received bacterial Broccoli from Addgene.

Week 7 (160710 – 160716)

Flow Cytometry analysis of Interlab constructs using absolute bead calibration.

Designed primers to switch out RBS on K2066014 with commonly used RBSes from community collection.

Moved Broccoli onto UNS backbone

Perform a promoter swap Gibson assembly on K2066034 (switching to J23100, J23115), with and without RiboJ.

Created a version of K2066034 with broccoli aptamer.

Week 8 (160710 – 160716)

Measured fluorescence of DFHBI activated K2066041 and K2066042 (Broccoli parts), results were discouraging.

Retried assembly of Broccoli into K2066034.

Redesigned primers to move pTet GFP and TetR into UNS Backbones and combine pTet.

GFP and TetR into one plasmid, both with and without UNS sequences.

Performed RBS swaps using Gibson assembly on K2066014.

iGEM Mid-Atlantic meetup at UMD.

Week 9 (160724 – 160730)

Prepared Experiment video.

Worked on optimization of flow cytometry measurements, RBS swaps repeatedly giving low quality readings.

Cotransformation of K2066046 and K2066048.

Inductions of pACT-Tet + 57S cotransformations in 3.300LG.

Continued to troubleshoot Broccoli activation.

Used Gibson assembly to swap the T7 promoter into K2066034 and K2066051 with and without RiboJ.

Assembled K2066054, K2066055 using Gibson Assembly.

Performed backbone swaps on pACT-Tet and 52S + DT.

Week 10 (160731 – 160805)

Assembled K2066053 using Gibson Assembly.

Continued troubleshooting bad flow cytometry readings for inductions.

Week 11 (160806 – 160812)

Performed Promoter swap Gibson Assemblies on K2066035 for all promoter J23100-J23119, R0040, R0010, R0011, J23150, J23151, w/ and w/out RiboJ.

Cotransformation of 85x TetO Array and pTet GFP + TetR.

Week 12 (160813 – 160819)

Continued cloning pipeline for promoter swap Gibsons from last week.

Performed various cotransformations of synthetic enhancer parts to test induction.

Tried new permutations of broth and strain with pTet-GFP + TetR cotransformations to troubleshoot inductions.

Induced 52S + pACT-Tet, 55AS + pACT-Tet, and 52S DT UNS + pACT-Tet (low copy) cotransformations.

Redid promoter swap assemblies from Week 11 that were sequence disconfirmed.

Week 13 (160820 – 160826)

Continued cloning pipeline on promoter swap assembly redo’s.

Performed backbone swap on K2066053 and 85x TetO array.

Perform inductions on K2066053 with and without 85x TetO cotransformation.

Redid Interlab measurements with improved flow cytometry protocol, started from plates of glycerol streak outs.

Performed (co)transformation of K2066053 with and without 85x TetO into BL21.

Week 14 (160827 – 160902)

Performed 14 step inductions on K2066053 with 85x TetO.

Designed primers to allow insertion of LacO and TetO arrays into BioBrick compatible Backbones.

Decided to switch to a weaker RBS driving expression of TetR on K2066053.

Completed raw data collection for interlab devices!

Gibson assembly to combine pLac sfGFP with constitutive LacI.

Week 15 (160903 – 160909)

aTc inductions K2066109 +/- 85x TetO Binding Array.

Retry to cut and ligate LacO and TetO arrays into UNS backbone.

Arabinose induction of K2066028 and K2066029.

Week 16 (160910 – 160916)

Attempt to make K2066111 from K2066110 using Gibson assembly.

Swap mCherry and sfGFP in 52S using Gibson Assembly.

Gibson assembly to move 55AS onto UNS backbone.

BL21 transformations of K2066014, K2066028 + K2066014 (low copy), pK2066023 (low copy), K2066029 + K2066023 (low copy), K2066025 + k2066016 (low copy) (to be induced).

Transform K2066109 + K2066011 and K2066053 + K2066011 for induction.

Gibson assembly to add DT to UNs NRII and UNS 55AS.

Transform K2066110 and K2066111 with and without LacO array in 10Beta.

Week 17 (160917 – 160923)

Induce K2066110 and K2066111 with and without LacO array.

Test known-concentration GFP with plate reader.

Gibson assembly to move K2066028 and K2066029 onto 3K3 backbone.

Transform K2066028 and K2066029 into 5alpha.

Transform K2066110 and K2066111 with and without LacO array in BL21.

Transform combinations of OA pACT tet, UNS 52S, UNS55, and sfGFP in BL21.

Present research at Family Weekend Biology Seminar.

Week 18 (160924 – 160930)

Gibson assembly to move the synthetic enhancer parts to 3k3 (52s, 55as, 52s+NRII, 55as+sfgfp).

Induce K2066110 and K2066111 with and without LacO array with IPTG.

Flow Cytometry analysis of UNS NRII 52S (1C3) + TetR (3K3), K2066110 and K2066111 with LacO array.

Induce pACT-Tet (OA) + UNS 52S+DT, pACT-Tet (OA) + UNS 55AS+DT, and pACT-Tet (OA) + UNS 55AS sfGFP with aTc and perform Flow Cytometry analysis.

Gibson assembly to move K2066025 on 1C3 backbone.

Transform combinations of synthetic enhancer parts, all failed.

Gibson assembly to move 55AS sfGFP NRII onto 3K3 backbone.

Week 19 (161001-161007)

Induce K2066110 with IPTG and measure with flow cytometry.

Induce 55as 3k3 +pACT Tet OA, 55as sfgfp 3k3 +pACT Tet OA, 55as NRII 1C3 + tetR 3k3, 52s NRII 1C3 + tetR 3k3, and 55as NRII sfgfp 1C3 + tetR 3k3 with aTc and measure with flow cytometry.

Try to transform combinations of synthetic enhancer parts again in LG 3.300 Induced with aTC and constant IPTG constructs that grew and measured with flow cytometry until the device failed.

Measured remaining induced constructs with plate reader.

Gibson assembly to put tetR on 55AS sfGFP NRII 1C3 and 52S NRII sfGFP 3K3.

Transform 52S (OA) + pACT-Tet (OA), with and without 85x tetO, in LG3.300, as well as K2066053 and K2066053 + 85x tetO in BL21 .

Plated out the 52S + pact-Tet glycerol stock which worked.

Week 20 (161008-161014)

Test known-concentration GFP on plate reader again to get instrument variability.

Induce with aTC 52S OA + pACT-Tet OA + 85x tetO 1C3.

Measure induced construct in plate reader.

Gibson assembly to move K2066029 onto 1A3 and to assemble K2066025.

Induce 53 1C3 + 85x tetO 1A3 with aTC.

Transform K2066025 to measure K2066025 induction in BL21 with K2066016 3K3 to get RiboJ measurements.

Transform K2066110 and K2066110 + lacO array to characterize the pJD100 48x lacO array.

Transform K2066029 in BL21 and 5alpha strains.

Induce 52S OA + pACT-Tet OA LG3.300 with aTC.

Measured recent induced constructs with plate reader.

Induced K2066110 with IPTG, K2066110 + lacO array with IPTG, K2066029 with arabinose, and K2066014 with K2066016 with IPTG and measured on plate reader.

Explicitly attempt to replicate 52S measurements from Amit et al. (Synthetic Enhancer...).

Gibson assembly to move K2066029 1C3 onto 1A3 backbone and to move K2066023 (pTet GFP, K1493504) onto 1A3 backbone (no UNS)..

Transform previous Gibson assemblies and K2066053 with and without 85x tetO on 1C3 and 1K3.

Transform 52S + NRII 1K3 AND tetR 1A3, 52S sfGFP + NRII 3K3 AND tetR 1A3, 52S tetR sfGFP + NRII 3K3, 52S tetR sfGFP + NRII 3K3 AND 85x tetO 1C3, and 52S OA Kan AND pACT-Tet OA Amp AND 85x tetO 1C3.

Week 21 (161015-161019)

Induce transformations, 52S OA Kan AND pACT-Tet OA Amp LG3.300, K2066053 1C3 + 85x tetO 1K3 BL21, and K2066053 1A3 + 85x tetO 1C3 BL21.

Cotransform K2066025 1C3 and all other RiboJ parts with K2066016 3K3 in BL21 to get induction curve.

Induced and measure recently transformed synthetic enhancer constructs.

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