We found that we may not need to order any BioBricks, as Lethbridge only used the sequences of the parts, and then proceeded synthesized them. We decided that we want
some more time to determine what we need to send for synthesis, as we want to be exact with what we need. We also decided some different ways to continue the second construct, which Lethbridge never got to test. We want to add purification tags to both construct one (clotting blood) and construct two (localizing the clot). We could use Histags, and in construct two we need Factor 13, an antimicrobial peptide.
Came up with ideas and people to contact
We began looking at places/people we could contact who we can ask for more insight into potential applications for our project. We sent an email to Dr. Lavik, who is
knowledgeable about using nanoparticles to increase the rate of coagulation and lessen the possibility of infection and inflammation. We also talked a little bit about a potential way to test the effectiveness of the enzymes we produce with our constructs. We also introduced the idea of potentially adding a numbing agent into the construct.
Did research to figure out how to best design constructs
Finished calibration protocol for absorbance and fluorescence to obtain needed measurement parameters for our experiment
Absorbance settings:
Fluorescence settings:
Complete protocol for absorbance/fluorescence for parts 1-6 (not controls) and recorded data
We finished all calculations for LB+Chlor media
Finished taking absorbance and fluorescence of time point 0 hours data
We made 70% alcohol
Labeled and prepped plates and tubes for CFU protocol
Completing dilutions for protocol and then needs to plate
.1 dilution was not precise, here is the absorbance data from that (measuring 550ABS)
LWe checked the sequence of the TPA we amplified by aligning it to the biobrick parts sequence.
We used Clustal Omega to do this.
We checked from around 580 bp to the end and everything was great (almost perfect matches).
We also checked the quality of the sequence using a chromatography viewer. (Quality was very good)
Next Step: Will request that VF2 sample be resequenced
Areas to look more into:
Learned that tPA is very potent and only used as a treatment in high-risk/severe stroke cases. What makes it so potent? Is there a way to modify that aspect?
Methods of delivery for tPA? Literature shows some use for targeted treatment of blood clots. (Reference: https://www.ncbi.nlm.nih.gov/pubmed/28796540)
Plasmid prep with bacterial biobrick plasmid backbone BBa-I746909 (906 bp) using Monarch Miniprep kit. We obtained plasmid concentration and purity using the Nanodrop.
Ran it through gel Gene designing (shorter TPA: reteplase) for optimized expression in E. Coli Changing codons from mammalian preference to codons based on E.Coli codon preference. Lisa ordered three constructs for us:
Started to work with assays we created one that had the following materials DPBS,Trypsin and Acetate. With these materials we did the following
Performed colony PCR screening, with a couple potential positives.
Miniprepped genes and sent them to Johns Hopkins for screening
set up four ligations:
Redigested and ligated g-blocks
Observations of plates:
Colony PCR screen to verify that insert is present
Colony PCR screen https://www.neb.com/protocols/2012/09/06/protocol-for-onetaq-2x-master-mix-with-standard-buffer-m0482 There were many colonies on Reteplase today. Observations:
Colony PCR screen to check for insert
Screened colonies with colony PCR
Various positive samples that need to be sequenced to determine whether they’re our samples or not
looked at sequencing results
3 reactions -- T7-TPA, weak-TPA, reteplase
Conducted PCR Screening on our next set of potential positives and sent the rear third of construct to be sequenced
No colonies found on reteplase 16 colonies on weakpromoter-TPA 10 colonies on T7-TPA check for insert using colony PCR screen
Prepped for a His Tag Screening. Decided to use samples 4-3 and 1-5 (and some of 1-4). Didn’t finish.
Performed PCR cleanup of 9 colonies
Concentrated samples using NanoSep
Finished His Tag Screening and ran a protein gel.
