Difference between revisions of "Team:BroadRun-NorthernVA/Notebook"

Lab Notebook

Welcome to our Lab Notebook! Here, we have documented the work done in our project so we can see and keep track of how our project is progressing.

June

August 8

• Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The amylase gene used for all three constructs was the alpha amylase coding sequence from Bacillus amyloliquefaciens. The Genbank accession number is J01542.1. First parts used were pCyc (medium) promoter (Part BBa_I766555), Kozak sequence (Part BBa_K165002), Alpha amylase coding sequence (Genbank Accession #J01542.1), and ADH1 Terminator (Part BBa_K801012).The alpha amylase coding sequence contained an EcoR1 restriction site, so GeneDesign was used to eliminate the site and optimize the construct for S.cerevisiae. However with this combination, IDT would not accept our design, due to repeats and sections with a low GC count. The constructs were redesigned, keeping the same coding sequence and Kozak sequence, but using the minimal cyc promoter (Part ) and minimal adh1 terminator (Part ). In order to test several variants on the expression of amylase, two promoters, and two secretion sequences were used. Plasma DNA was used to map restriction sites, and Gene design used to remove restriction sites. The final makeup of the gene constructs are listed below.

  Construct 1

• Biobrick prefix
• Promoterless
• Kozak sequence (Part BBa_K165002)
• Native secretion sequence, from Bacillus amyloliquefaciens
• Alpha amylase coding sequence from Bacillus amyloliquefaciens
• ADH1 Terminator (Part BBa_K392003)
• Biobrick Suffix

Construct 2

• Biobrick prefix
• Minimal cyc promoter (Part BBa_K105027)
• Kozak sequence (Part BBa_K165002)
• Native secretion sequence, from Bacillus amyloliquefaciens
• Alpha amylase coding sequence from Bacillus amyloliquefaciens
• ADH1 Terminator (Part BBa_K392003)
• Biobrick Suffix

Construct 3

• Biobrick prefix
• Minimal cyc promoter (Part BBa_K105027)
• Kozak sequence (Part BBa_K165002)
• Native secretion sequence, from Bacillus amyloliquefaciens
• Alpha amylase coding sequence from Bacillus amyloliquefaciens
• ADH1 Terminator (Part BBa_K392003)
• Biobrick Suffix

Week 2

Wet Lab

• Isolated DNA from the transformed plasmids, for use in PCR later in the week.
• PCR used to amplify and isolate our desired DNA from their plasmids, using the plasmids we designed in week one.
• PCR products then run on a gel to confirm they were of the correct size. Desired DNA was isolated for later use using gel extraction.
• Primers designed for GvpA , GvpC, and for the purposes of isolating FliC from the E. Coli genome. This will be used later on in the project to study the effect of the flagella in the presence of gas vesicles.
• Bacteria transformed with plasmids taken from the distribution. The parts used contained useful things such as GFP, a promoter, and vectors that will be used at a later stage. The useful DNA from these transformations was isolated and digested using EcoR1 and Pst1, to ensure the sizes were correct.
• One transformation was done using 2 different vectors containing RFP (pSB6A1 and pSB3C5), and two plasmids containing GFP, each with a different RBS. Another used instead the vector pSB1C3 and another containing the promoter J23100(CHECK) – both expressing RFP. This later transformation will be run on a gel in week 3.
• We had chosen 2 samples of our miniprepped DNA from out first transformation (one with GvpA, the other with GvpC) to be sent off for sequencing. At the end of the week, this sequenced data was analysed, and proved that out isolated DNA contained the desired sequences.
• An important step was taken this week: the ligation of our PCR-produced GvpA and GvpC - containing prefix, suffix and desired RBS – into separate pSB1C3 plasmid vectors. In order to do this, the PCR products and the vector underwent a restriction digest using EcoR1 and Pst1. The resultant DNA fragments were run on a gel and the fragments containing GvpA and GvpC and pSB1C3 were cut out and the DNA purified using gel extraction. GvpA was then ligated into pSB1C3. Seperately, GvpC was ligated into pSB1C3. The pSB1C3 was obtained by digesting the GFP-0034 BioBrick part from the distribution.
• As the above was taking place, FliC was also digested with EcoR1 and Pst1. The resultant gel showed a restriction site within FliC – a problem which must be resolved.